Abstracts From The World Federation of Neuro-Oncology Second Quadrennial Meeting and The Sixth Meeting of The European Association For Neuro-Oncology

Abstracts from the
World Federation of Neuro-Oncology

Second Quadrennial Meeting
and the
Sixth Meeting of the
European Association for Neuro-Oncology
May 58, 2005
Edinburgh, United Kingdom
Copyright 2005 by the Society for Neuro-Oncology
Neuro-Oncology J ULY 2005 281
1. EXTENDED ABSTRACT: ADVANCES IN DIAGNOSIS OF
SUPRATENTORIAL LOW- GRADE GLIOMAS IN ADULTS
J.-Y. Delattre; Hpital de la Salptrire, Paris, France
In more than 80% of cases, the rst manifestation of low-grade glioma
(LGG) is a partial or (secondarily) generalized seizure in a young adult
(median age, 3539 years) who is otherwise in good general health with a
normal neurological examination. Much more rarely, progressive decit,
cognitive dysfunction, or intracranial hypertension will reveal the disease.
In this setting, MRI of the brain with and without gadolinium infusion is
mandatory. The MRI aspect is not specic, but the diagnosis is strongly
suspected when a nonenhancing mass (hypointense on T1-weighted images
and hyperintense on T2-weighted images or FLAIR sequences) is discov-
ered in the frontal-temporal-insular region or within the parasagittal
frontal-parietal regions, the most frequent and characteristic locations of
LGG (Duffau and Capelle, 2004). Occasionally, contrast enhancement is
seen, or the tumor widely inltrates the brain at the onset (gliomatosis cer-
ebri). MR spectroscopy and PET scan (uorodeoxyglucose and methionin)
are useful to guide a biopsy and for differential diagnosis. The most fre-
quently considered differential diagnosis includes encephalitis in young
patients and stroke in older patients (Calli et al., 2002). Denite diagno-
sis eventually relies on microscopic and genetic examination of a tumor
sample. Microscopic examination is crucial to exclude tumors that can be
cured by surgery alone, such as dysembryoplasic neuroepithelial tumor,
ganglioglioma, or even pilocytic astrocytomas, which are not considered
here, and to detect features of anaplasia, a common nding in apparently
benign tumors on MRI (Scott et al., 2002). The value of pathological
examination to precisely identify the tumor cell type is more debatable.
Indeed, the WHO classication of LGGs (Kleihues, 2000) relies on mor-
phological criteria to dene astrocytomas, oligodendrogliomas, or mixed
gliomas. This classication remains imperfect because of incomplete repro-
ducibility, even for a single observer, and lack of specic marker of tumor
subtype (Mokhtari et al., 2005). This fact is illustrated by a dramatic rise
in the incidence of oligodendrogliomas in many centers over the last decade
associated with a concomitant reduction of astrocytomas, even if the diag-
nostic criteria did not change signicantly during this period (Burger, 2002,
and data not shown). As a consequence, the respective frequency of tumor
subtypes varies considerably among institutions, a feature with practi-
cal implications because evidence suggests that the management of these
tumors should be tailored according to the main tumor cell type (Hoang-
Xuan et al., 2004).
Molecular classication is an important adjunct to the classication
of LGG. Up to 75% to 80% of LGGs have one of the two key early
genetic alterations reported in gliomas, including chromosome 1p loss or
P53 expression (which is linked to P53 mutation), and 20% to 25% have
both alterations or neither. Overall, these two molecular alterations are
strongly mutually exclusive. Tumors with 1p loss almost always have a
morphological pattern of oligodendrogliomas (honeycomb appearance)
or rarely a mixed glioma pattern (Reifenberger et al., 1994). On the other
hand, tumors with P53 expression are more heterogeneous: 50% of them
are brillary astrocytomas, but the other half have either an oligodendrogli-
oma or a mixed glioma pattern. Thus, an astrocytoma pattern is practically
never associated with 1p loss alone, but an oligodendroglioma or mixed
glioma pattern may be associated with either 1p loss or P53 expression.
There is a correlation between the prole of molecular alterations and
tumor location (Laigle-Donadey et al., 2004; Zlatescu et al., 2001). Chro-
mosome 1pdeleted tumors are preferentially located in the frontal regions
and frequently infiltrate widely the parenchyma, and P53-expressing
tumors are mainly located in the parietal-temporal-occipital regions and
are more circumscribed on MRI. Interestingly, a correlation between P53
mutation and preferential topography of the tumor has also been noted in
other tumors, such as gastric or colon cancers.
Knowledge of the genetic status of LGGs has important consequences
because chromosome 1p loss seems to be one of the most important favo-
rable prognostic factors in LGG, possibly surpassing classic factors such as
age, performance status, histological subtype, or tumor size at diagnosis.
Furthermore, 1p-deleted LGGs are chemosensitive in half of the cases, but
1p-intact tumors much more rarely respond to chemotherapy (Hoang-Xuan
et al., 2004).
Since a biopsy does not necessarily encompass the most aggressive
part of a tumor, microscopic examination of a small specimen taken at
the periphery of the lesion may be falsely reassuring when it detects only
features of LGG. In these circumstances, detection of the so-called late
genetic alterations of gliomas, particularly the ominous combination of
EGFR amplication with chromosome 10q loss, may alert the clinician that
the underlying tumor is much more malignant than expected by histology.
The natural history of LGG is changing, with a striking increase
of survival over the last two decades. This feature reects the fact that
the diagnosis is made much earlier than before and underlines the need
for a careful evaluation of the risk/benet ratio before administration of
potentially toxic treatment. An important observation is that LGGs grow
inexorably over time during the period, which often lasts many years, pre-
ceding the malignant transformation and terminal phase of an LGG course
(Mandonnet et al., 2003), a feature pleading for early surgery when it is
possible.
References
Burger, P.C. (2002) What is an oligodendroglioma? Brain Pathol. 12,
257259.
Calli, C., Ozel, A.A., Savas, R., Kitis, O., Yunten, N., and Sener, R.N
(2002) Proton MR spectroscopy in the diagnosis and differentiation of
encephalitis from other mimicking lesions. J. Neuroradiol. 29, 2328.
Duffau, H., and Capelle, L. (2004) Preferential brain locations of low-grade
gliomas. Cancer 100, 26222626.
Hoang-Xuan, K., Capelle, L., Kujas, M., Taillibert, S., Duffau, H., Lejeune,
J., Polivka, M., Criniere, E., Marie, Y., Mokhtari, K., Carpentier, A.F.,
Laigle, F., Simon, J.M., Cornu, P., Broet, P., Sanson, M., and Delattre,
J.Y. (2004) Temozolomide as initial treatment for adults with low-grade
oligodendrogliomas or oligoastrocytomas and correlation with chro-
mosome 1p deletions. J. Clin. Oncol. 22, 31333138.
Kleihues, P. (2000) Pathology and Genetics: Tumours of the Nervous Sys-
tem. WHO Classication of Tumours. Lyon: IARC Press.
Laigle-Donadey, F., Martin-Duverneuil, N., Lejeune, J., Criniere, E.,
Capelle, L., Duffau, H., Cornu, P., Broet, P., Kujas, M., Mokhtari, K.,
Carpentier, A., Sanson, M., Hoang-Xuan, K., Thillet, J., and Delattre,
J.Y. (2004) Correlations between molecular prole and radiologic pat-
tern in oligodendroglial tumors. Neurology 63, 23602362.
Mandonnet, E., Delattre, J.Y., Tanguy, M.L., Swanson K.R., Carpentier,
A.F., Duffau, H., Cornu, P., Van Effenterre, R., Alvord, E.C., Jr., and
Capelle, L. (2003) Continuous growth of mean tumor diameter in a
subset of grade II gliomas. Ann. Neurol. 53, 524528.
Mokhtari, K., Paris, S., Aguirre-Cruz, L., Privat, N., Criniere, E., Marie,
Y., Hauw, J.J., Kujas, M., Rowitch, D., Hoang-Xuan, K., Delattre,
J.Y., and Sanson, M. (2005) Olig2 expression, GFAP, p53 and 1p loss
analysis contribute to glioma subclassication. Neuropathol. Appl.
Neurobiol. 31, 6269.
Reifenberger, J., Reifenberger, G., Liu, L., James, C.D., Wechsler, W.,
and Collins, V.P. (1994) Molecular genetic analysis of oligodendro-
glial tumors shows preferential allelic deletions on 19q and 1p. Am. J.
Pathol. 145, 11751190.
Scott, J.N., Brasher, P.M., Sevick, R.J., Rewcastle, N.B., and Forsyth, P.A.
(2002) How often are nonenhancing supratentorial gliomas malignant?
A population study. Neurology 59, 947949.
Zlatescu, M.C., TehraniYazdi, A., Sasaki, H., Megyesi, J.F., Betensky,
R.A., Louis, D.N., Cairncross, J.G. (2001) Tumor location and growth
pattern correlate with genetic signature in oligodendroglial neoplasms.
Cancer Res. 61, 67136715.
2. EXTENDED ABSTRACT: PROSPECTIVE CLINICAL
TRIALS OF ADULT-SUPRATENTORIAL LOW- GRADE
GLIOMA
E.G. Shaw; Department of Radiation Oncology, Comprehensive Cancer
Center, Brain Tumor Center of Excellence, Wake Forest University
School of Medicine, Wake Forest, NC, USA
There are three current controversies in the radiotherapeutic man-
agement of adults with supratentorial low-grade glioma (LGG) based on
numerous retrospective studies that have been published in the medical
literature. The rst is the optimum timing of radiation therapy (RT). Fol-
lowing maximum surgical resection, should RT be given immediately post-
operatively, or is surgery alone adequate treatment, with RT added only if
imaging progression occurs? Second, if RT is to be given, is there an advan-
tage in terms of local control and survival for higher rather than moderate
to lower doses of RT? Third, given the modest cure rates of surgery with or
Abstracts*
*Authors whose names appear in italic are the presenting authors.
Neuro-Oncology
Abstracts from the World Federation of Neuro-Oncology Meeting
282 Neuro-Oncology J ULY 2005
without RT, will disease-free and overall survival be improved by adding
chemotherapy to RT? Four prospective randomized clinical trials in adults
with supratentorial LGG were conducted in the United States, Canada, and
Europe during the 1980s and 1990s that addressed these questions (Eyre
et al., 1993; Karim et al., 1996, 2002; Shaw et al., 2002; van den Bent et
al., 2004). The data from the nearly 1000 patients treated in these studies
are summarized in this abstract. In addition, the schemata from recently
completed and planned phase 2 and phase 3 clinical trials in the United
States and Europe will be reviewed.
Summary of Published Clinical Trials: The ve-year overall survival
and progression-free survival rates in these four published studies ranged
from 58% to 72% and from 37% to 55%, respectively. European Organiza-
tion for Research and Treatment of Cancer (EORTC) Study 22845 random-
ized 311 adults to postoperative observation or RT. There was no difference
in the ve-year survival rate between the two arms. Irradiated patients had
a signicantly improved ve-year progression-free survival rate. EORTC
Study 22844 randomized 379 adults to low- versus high-dose RT. Similarly,
an intergroup study conducted by the North Central Cancer Treatment
Group, the Radiation Therapy Oncology Group (RTOG), and the Eastern
Cooperative Group randomized 211 adults to low- versus high-dose RT.
There was no difference in the ve-year overall or progression-free survival
rates between the two dose groups in either study. A Southwest Oncology
Group study randomized 60 adults with incompletely resected LGG to RT
alone or RT plus lomustine (CCNU) chemotherapy. There was no difference
in outcome between the two treatment arms. Important prognostic factors
for overall survival in these trials include extent of surgical resection, histol-
ogy, tumor size, and age.
Summary of Recently Completed and Planned Clinical Trials: The
RTOG recently completed Study 9802, in which adults with supratento-
rial LGG were placed into risk groups and treated accordingly. Low-risk
patients, dened as those younger than 40 years and who underwent gross
total resection were observed postoperatively. The preliminary data from
111 low-risk patients will be presented at this meeting (see Shaw et al.,
2002). High-risk patients in RTOG 9802, dened as those aged 40 years
or older or who underwent subtotal resection or biopsy, were randomized
to RT alone (54Gy to tumor with a 2cm margin) or RT followed by six
cycles of PCV chemotherapy (procarbazine, CCNU, and vincristine). It will
be several years before data from the high-risk group will be available.
The RTOG has just opened a randomized phase 2 study of RT (54Gy) fol-
lowed by temozolomide chemotherapy, or temozolomide both during and
following RT. The Eastern Cooperative Oncology Group has just submit-
ted a concept to the National Cancer Institute for a phase 3 clinical trial
in high-risk adults with supratentorial LGG, randomizing them to RT
alone (50.4Gy to tumor with a 2cm margin) or RT followed by six cycles
of temozolomide chemotherapy. The EORTC is planning a phase 3 clinical
trial in adults with supratentorial LGG, randomizing them to RT (50.4Gy)
or temozolomide (no RT), stratifying patients by chromosome 1p deletion
status (present versus absent) (Dr. Martin van den Bent, personal commu-
nication, November 30, 2004).
Conclusions: Based on the information presented, the following con-
clusions can be made: In adults with LGG, there is no difference in overall
survival whether RT is given postoperatively or delayed to the time of recur-
rence. However, about two-thirds of adults with LGG will develop tumor
progression by ve years following surgery alone. When RT is adminis-
tered, lower doses produce a similar survival outcome as higher doses with
less neurotoxicity. Data on whether chemotherapy (PCV or temozolomide)
either alone or with RT improves outcome is forthcoming from recently
completed or ongoing prospective clinical trials.
References
Eyre, H.J., Crowley, J.J., Townsend, J.J., et al. (1993) A randomized trial of
radiotherapy versus radiotherapy plus CCNU for incompletely resected
low-grade gliomas: A Southwest Oncology Group study. J. Neurosurg.
78, 909914.
Karim, A.B.M.F., Maat, B., Hatlevoll, R., et al. (1996) A randomized trial
on dose-response in radiation therapy of low-grade cerebral glioma:
European Organization for Research and Treatment of Cancer Study
(EORTC) Study 22844. Int. J. Radiation Oncology Biol. Phys. 36,
549556.
Karim, A.B.M.F., Afra, D., Cornu P., et al. (2002) Randomized trial on
the efcacy of radiotherapy for cerebral low-grade glioma in the adult:
European Organization for Research and Treatment of Cancer Study
22845 with the Medical Research Council Study BR04: An Interim
Analysis. Int. J. Radiation Oncology Biol. Phys. 52, 316324.
Shaw, E., Arusell, R., Scheithauer, B., et al. (2002) Prospective randomized
trial of low- versus high-dose radiation therapy in adults with supraten-
torial low-grade glioma: Initial report of a North Cancer Central Treat-
ment Group / Radiation Therapy Oncology Group / Eastern Coopera-
tive Oncology Group Study. J. Clin. Oncol. 20, 22672276.
van den Bent, M.J., Afra, D., de Witte, O., et al. (2004) Long term results
of EORTC 22845/MRC BR4: A randomized trial on the efcacy of
radiation therapy in adult low-grade glioma (abstract). Neuro-Oncol-
ogy 6, 364.
4. EXTENDED ABSTRACT: MOLECULAR CYTOGENETIC
TESTING IN NEUROONCOLOGY
R. Jenkins; Mayo Clinic and Foundation, Rochester, MN, USA
Testing for genetics abnormalities in gliomas is rapidly becoming a part
of clinical neuro-oncology. Combined 1p/19q deletion; EGFR amplica-
tion, mutation, or vIII overexpression; and MGMT methylation or under-
expression are just the rst of many genetic markers that have or will be
part of routine clinical practice. This presentation will focus on the discov-
ery, validation (clinical and analytic), development, and implementation
of molecular cytogenetic tests in gliomas, with emphasis on 1p and 19q
deletion testing.
Combined 1p and 19q deletion is associated with gliomas of oligo-
dendroglial lineage. In addition, several retrospective studies suggest that
patients whose glioma has 1p and 19q deletion have a better survival and
a better response to chemo- and/or radiation therapy. Recently, Cairncross
and colleagues reported preliminary analyses of RTOG 9402 (Cairncross
et al., 2004). RTOG 9402 was a randomized trial to determine if dose-
intense treatment with PCV (procarbazine, lomustine, and vincristine)
before radiation therapy prolongs overall survival (primary endpoint) or
progression-free survival (secondary endpoint) versus radiation therapy
alone in anaplastic oligodendrogliomas and mixed oligoastrocytomas.
Patients whose tumor had 1p and 19q deletion had a signicantly better
overall survival and a signicantly longer time to progression when treated
with PCV. These retrospective and prospective studies have spurred the
development of clinical 1p and 19q deletion testing. Fluorescence in situ
hybridization (FISH) 1p and 19q deletion testing was formally offered for
Mayo Clinic patients (and for other patients through the Mayo Medical
Laboratories) during the fall of 2002. Since the introduction of the clinical
test, we have performed approximately 300 tests per year. The presentation
will describe the implementation of the FISH 1p and 19q deletion test and
our current experience using this test.
Because of the clinical signicance of 1p and 19q deletions in gliomas,
there is strong interest in the identication of the relevant gene (or genes)
on these chromosome arms. We are following multiple strategies to identify
the target genes, including deletion mapping in primary tumors and cell
lines, somatic cell hybrid analyses, comprehensive expression analysis, and
genetic association studies. The presentation will review the current status
of these 1p and 19q gene identication experiments.
Reference
Cairncross, J.G., et al. (2004) ASCO Annual Meeting Proceedings 2004.
J. Clin. Oncol. 22S, 1500.
5. EXTENDED ABSTRACT: OVERVIEW AND CURRENT
STATUS OF BRAIN TUMOR EPIDEMIOLOGY
M. Wrensch; Department of Neurological Surgery, University of
California, San Francisco, CA, USA
Epidemiology aims to describe the occurrence of a disease in place
and time, understand and explain the distribution and causes of the dis-
ease, and address factors that inuence survival after diagnosis. Major
challenges in conducting epidemiologic studies of brain tumors include
(1) substantial histologic and molecular heterogeneity of tumors between
and within histologic groups, (2) the relative rarity of any given subtype,
(3) geographic and temporal heterogeneity in reporting requirements and
classication systems, (4) a paucity of well-established risk factors, and
(5) the rapid fatality associated with the most common and lethal form
of primary malignant brain tumors, glioblastoma multiforme, necessitat-
ing studies that often involve the use of proxy respondents for gathering
potentially relevant life-history information. To meet these challenges,
recent descriptive epidemiologic studies of primary brain tumors highlight
strengths and weaknesses in population data to encourage consistent and
reliable classication of primary brain tumors to enable meaningful track-
ing of temporal and geographic variation. For example, the Central Brain
Tumor Registry of the United States (CBTRUS, www.cbtrus.org) provides
a resource for gathering and disseminating current epidemiologic data
on all primary brain tumors, for the purposes of accurately describing
their incidence and survival patterns, evaluating diagnosis and treatment,
facilitating etiologic studies, establishing awareness of the disease, and ulti-
mately, for the prevention of all brain tumors. Etiologic and prognostic
studies of brain tumors increasingly require multidisciplinary collabora-
tion between surgeons, neuro-oncologists, geneticists, toxicologists, epide-
miologists, and molecular and environmental scientists. Molecular tumor
markers are being identied that predict survival and treatment response
with hope of even greater gains in this area with emerging array technolo-
gies. Regarding risk factors, studies of inherited susceptibility and constitu-
tive polymorphisms in genes pertinent to carcinogenesis (e.g., DNA repair,
detoxication, and immune function genes and mutagen sensitivity) have
revealed provocative ndings. Consistent inverse associations of history of
allergies with glioma risk observed in several large studies suggest a pos-
sible role of immune factors in gliomagenesis or progression. Studies also
continue to suggest that brain tumors might result from workplace, dietary,
and other personal and residential exposures. Recent studies of cell phone
use have suggested that acoustic neuromas, but not other primary brain
tumors, might be inuenced by cell phone use. Only a small proportion
of primary brain tumors are attributable to proven and widely accepted
causes of brain tumors (i.e., rare hereditary syndromes, therapeutic radia-
tion, and immune suppression giving rise to brain lymphomas), suggesting
the need for further discovery. Progress in understanding primary brain
tumors will require large studies of well-dened histologic and molecular
tumor types incorporating assessment of potentially relevant information
on subject susceptibility and environmental and noninherited endogenous
factors (viruses, radiation, and carcinogenic or protective chemical expo-
sures through diet, workplace, oxidative metabolism, or other sources). To
facilitate this progress, in February 2003, researchers and advocates formed
the Brain Tumor Epidemiology Consortium (BTEC) (20042006 European
Co-Chairs, Beatrice Malmer, Sweden, and Siegal Sadetzki, Israel, and
United States Co-Chairs, Faith Davis, Illinois, and Melissa Bondy, Texas).
The National Cancer Institute provided support for the initial meeting,
and the National Brain Tumor Foundation, Pediatric Brain Tumor Founda-
tion, Preuss Foundation, and CBTRUS have supported additional meetings.
BTEC is a self-directing consortium committed to developing multicenter
interdisciplinary collaborations that will lead to a better understanding of
etiology, outcomes, and prevention of brain tumors. Current initiatives
include family studies and additional epidemiologic studies of childhood
brain tumors, adult glioma, and meningioma.
6. EXTENDED ABSTRACT: QUALITY OF LIFE IN BRAIN
TUMOR PATIENTS: MEASUREMENT AND SUPPORT
M.J.B. Taphoorn; Medical Centre Haaglanden, Department of
Neurology/Neuro-Oncology, The Hague, The Netherlands
The objective of any anticancer therapy extends well beyond prolonga-
tion of survival. Palliation of symptoms and maintenance or improvement
of quality of life are important goals of therapy. Thus, the benets of exist-
ing or new cancer treatments that maintain or extend survival time need
to be weighed against side effects and a possible decrease in the patients
health-related quality of life (HRQOL). Health-related quality of life has
become an increasingly important endpoint in cancer studies, next to
outcome measures such as overall and disease-free survival, and is most
relevant in patients who cannot be cured of their disease (Efcace and Bot-
tomley, 2003).
In brain tumor patients, palliating symptoms and maintaining or
improving HRQOL is particularly relevant for several reasons. First,
patients with primary brain tumors (mainly gliomas) or metastatic tumors
in the central nervous system have a dismal prognosis and cannot be cured
of their disease. Second, brain tumor patients not only have to cope with
clinical symptoms, such as motor decit and epilepsy, but they are usually
also confronted with a decline in cognitive and emotional functioning as a
result of cerebral disease. Third, side effects of treatment for brain tumors
may have an even further negative impact on cerebral functioning (Mey-
ers, 1997).
Measurement of HRQOL: Despite the specic relevance for measuring
HRQOL in brain tumor patients, the interest in HRQOL emerged relatively
late in this patient group compared with more common cancers such as
breast or lung cancer. This has to do with the low incidence of primary
brain tumors, a former therapeutic nihilism toward brain cancer, and the
fact that the subjective nature of HRQOL assessment may be problematic
in brain tumor patients with mental impairments.
Health-related quality of life is dened as a multidimensional con-
cept consisting of at least physical, psychological, and social phenomena.
Measuring outcome in terms of tumor size, time to tumor progression,
and overall survival is relatively simple compared with outcome measures
such as impairment, disability, or handicap, which require (symptom)
scales (Heimans and Taphoorn, 2002). These objective scales, however,
do not adequately measure the patients HRQOL. Health-related quality
of life is an even more complex outcome measure, demanding a multidi-
mensional instrument that should be lled out by the patient (self-report
questionnaire). Both generic and disease-specic questionnaires have been
developed and validated to assess HRQOL in cancer patients. To measure
HRQOL in brain tumor patients, the generic European Organization for
Treatment and Research of Cancer Quality of Life Questionnaire (EORTC
QLQ-C30) is used in combination with the Brain Cancer Module (BN 20)
or the Functional Assessment of Cancer Therapy (FACT) generic question-
naire together with the FACT brain module (Osaba et al., 1996; Weitzner
et al., 1995).
Health-Related Quality of Life in Brain Tumor Patients: HRQOL is
increasingly being measured as a secondary outcome in brain tumor trials
in which the efcacy of a (new) therapy is being evaluated. This holds true
for both low-grade and high-grade glioma patients.
Compliance with serial assessments of HRQOL is one of the major
problems in randomized trials. A low compliance with HRQOL assessment
is related to performance status and outcome and may cause a serious bias
in results (Roa et al., 2004; Walker et al., 2003).
Health-related quality of life in newly diagnosed high-grade glioma
patients appeared to be comparable to that in lung cancer patients and was
signicantly worse than that of healthy controls (Klein et al., 2001). In con-
trast, cognitive decit was far more pronounced in glioma patients than in
lung cancer patients, reecting the specic neurological decit. From stud-
ies in both newly diagnosed and recurrent high-grade glioma it is known
that HRQOL is related to disease burden and neurological decit (Osoba
et al., 1997).
In a comparison between low-dose and high-dose radiation schedules
in low-grade glioma patients, there appeared to be no difference in survival,
but HRQOL was more seriously impaired in the high-dose group than in
the low-dose group (Kiebert et al., 1998).
In two randomized trials the impact of temozolomide on HRQOL in
glioblastoma multiforme patients was investigated. No negative impact of
temozolomide on the patients HRQOL was observed, in contrast to the
toxic effect of procarbazine (Osoba et al., 2000a; Taphoorn et al., 2004).
Temozolomide even improved HRQOL in some of these patients, and this
was also observed in patients with recurrent anaplastic astrocytoma (Osoba
et al., 2000b).
Supportive Treatment: The brain tumor and its treatment may have
signicant physical, cognitive, emotional, and social effects on the patient.
The patients partner and family may also experience a negative emotional
and social impact. Apart from treatment of the tumor itself, supportive
treatment of the patient and the patients family may include medication
(antiepileptic drugs, steroids, antidepressants) and/or psychological and/or
cognitive support. As there is a steady increase in effective treatments for
brain tumor patients, the number of long-term survivors will grow. Long-
term negative effects of the tumor and its treatment demand an increasing
effort of doctors, nurses, and psychologists for supportive care (Remer and
Murphy, 2004).
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W.K.A., Brada, M., and Newlands, E. (1997) Effect of neurological
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7. PRELIMINARY RESULTS OF RTOG PROTOCOL 9802:
A PHASE II STUDY OF OBSERVATION IN COMPLETELY
RESECTED ADULT LOW- GRADE GLIOMA
E.G. Shaw,
1
Minhee Won,
2
David G. Brachman,
3
Christopher J.
Schultz,
4
John H. Suh,
5
Jan C. Buckner,
6
Geoffrey R. Barger,
7
Stephen
W. Coons,
8
Dennis E. Bullard,
9
and Minesh P. Mehta
10
;
1
Radiation
Oncology, Winston-Salem, North Carolina;
2
MA, RTOG, Philadelphia,
Pennsylvania;
3
Radiation Oncology, Phoenix, Arizona;
4
Radiation
Oncology, Milwaukee, Wisconsin;
5
Radiation Oncology, Cleveland,
Ohio;
6
Medical Oncology, Rochester, Minnesota;
7
Neuro-Oncology,
Detroit, Michigan;
8
Neuropathology, Phoenix, Arizona;
9
Neurosurgery,
Raleigh, North Carolina;
10
Human Oncology, Madison, Wisconsin; USA
In 1998, the RTOG initiated Protocol 9802 for adults with supratento-
rial low-grade glioma. Patients were divided into two groups based on risk.
Low risk was dened as age. These patients were observed postoperatively
with serial magnetic resonance imaging (MRI) scans and form the basis of
this report. Eligibility criteria included histologically proven WHO grade
II A, O, or OA based on central pathology review, age 18 and 60, Neu-
rologic Function Score (NFS) 3, supratentorial tumor location, GTR,
pre- and postoperative MRI scans available, and signed consent form. MRI
scans were obtained every 6 months. Prognostic factors analyzed for their
effect on overall survival (OS), progression-free survival (PFS), and tumor
recurrence included age (30 years), gender (male vs. female), KPS (90 vs.
100), NFS (0 vs. 13), histology (A astrocytoma or astrocytoma-dominant
OA vs. O oligodendroglioma or oligodendroglioma-dominant OA), con-
trast enhancement on pre-operative MRI scan (present vs. absent), pre-
operative tumor diameter (4cm), and baseline mini-mental status exam
score. Between 1998 and 2002, 116 patients were entered, 111 of whom
were analyzable. OS and PFS at 3 years for all patients was 97% and 73%,
respectively. The only two prognostic factors predicting for signicantly
poorer PFS in uni- and multivariate analyses were histology (univariate P
0.02, multivariate P 0.03, hazard ratio 2.33) and pre-operative tumor
diameter (univariate P 0.002, multivariate P 0.006, hazard ratio
2.90). The crude incidence of tumor recurrence was 38% for A vs. 20% for
O and 43% for tumors 4 cm vs. 18% for tumors 4 cm. The 3-year PFS
was 89% for O tumors 4 cm. An assessment of extent of surgical resection
based on the pre- vs. postoperative MRI scans is ongoing and will also be
analyzed as a possible prognostic factor. These data can be used to identify
low-risk low-grade glioma patients who may be candidates for postopera-
tive adjuvant treatment.
8. STUDY OF THE INDIVIDUAL CORTICAL
ORGANIZATION, CONNECTIVITY, AND PLASTICITY
APPLIED TO THE SURGERY OF LOW- GRADE GLIOMAS:
FUNCTIONAL AND ONCOLOGICAL RESULTS
H. Duffau,
1
L. Taillandier,
2
P. Gatignol,
1
M. Leroy,
1
E. Mandonnet,
1

D. Denvil,
1
M. Lopes,
1
M.C. Mitchell,
1
and L. Capelle
1
;
1
Hpital
Salptrire, Neurosurgery, Paris;
2
C.H.U. Nancy, Neurology, Nancy;
France
While considered by more and more authors, surgery of low-grade glio-
mas (LGG) remains matter of debate, because of (1) the risk for generating
sequelae (2) the impact, still controversial, of resection on the natural his-
tory of LGG. Taking account of the dynamic interactions between the brain
and LGG, we used functional mapping methods in order to study the indi-
vidual cortical organization, connectivity, and plasticity for each patient
harboring an LGG, and to apply these data to increase the benet/risk
ratio of surgery. From 1996 to 2004, 152 patients with no or only a slight
decit were operated on for a supratentorial LGG located near (57 cases)
or within (95 cases) eloquent areas. All the procedures were performed
by using intraoperative cortico-subcortical electrical stimulations, which
allowed tailoring of the resection according to functional boundaries. The
152 patients had neurological and neuropsychological assessment before
and after surgery. The quality of resection was systematically evaluated on
repeated post-operative MRI. Pre- and post-operative functional MRI was
performed in a subgroup of patients. Intraoperative study of the dynamic
organization of the functional networks was possible in all cases. Despite
a frequent immediate post-operative worsening, 94% of patients recovered
their preoperative status (or even improved) and returned to a normal socio-
professional life within 3 months after surgery. The resection was total
or subtotal in 88% of cases. With a median follow-up of 50 months, a
signicant statistical relationship was observed between the survival and
the quality of resection (P 0.02). Moreover, by comparing these results
with a similar surgical series of 100 LGGs operated on in the same institu-
tion without intraoperative functional mapping, we showed that electrical
stimulation allowed a signicant decrease of postoperative sequelae (5%
vs. 17%, P 0.002) and a signicant increase of the quality of resection
(51% vs. 25.4% of subtotal and 37% vs. 6% of total resections, P 0.001).
These ndings suggest a dynamic spatio-temporal functional organization,
with (a) before surgery, the recruitment of compensatory areas, explaining
the lack of decit despite the tumor growth in eloquent regions, (b) imme-
diately after surgery, the occurrence of a decit, likely due to the resection
of invaded areas participating (but not essential) to the function, and (c)
3 months after surgery, a recovery due to long-term functional reshaping.
The application of this brain dynamic potential can be considered to sig-
nicantly extend the limits of surgery in eloquent areas while minimizing
the risk of sequelae.
9. INTRAOPERATIVE LANGUAGE MAPPING IN
MULTILINGUAL PATIENTS WITH GLIOMAS
L. Bello, C. Giussani, F. Acerbi, M. Fava, V. Songa, P. Baratta, R.M.
Villani, and S.M. Gaini; Neurosurgery, University of Milano, Milano,
Italy
Surgical removal of lesions located close to or in areas of the brains
mediating speech requires the use of intraoperative techniques to localize
which cortical areas and subcortical tracts have those functions. Localiza-
tion of speech is particularly problematic in patients that are usually uent
with different languages. It has been reported that in bilingual patients
multiple and separate areas of the cortex mediate the different languages.
Other investigations pointed out that in bilingual patients common areas
of the brain are responsible for those functions. We report here ve cases
of patients harboring a left frontal glioma who were procient with three
to ve different languages in which a multiple language brain mapping was
undertaken during awake craniotomies for tumor removal. They were 3
males and 2 females, age ranging from 34 to 61 years. Language pro-
ciency was tested by submitting patients to confrontation tests for each
language during the pre-operative examination. Each language was tested
serially starting from the rst acquired language. Language mapping was
undertaken during asleep-awake craniotomy, by the use of an Ojemann
cortical stimulator and by using the largest current that did not produce
afterdischarge (from 3.5 to 6 mA) during counting and confrontation nam-
ing tests. Subcortical stimulation by using the same current threshold was
also applied during tumor resection, in a back and forth fashion. Our data
showed that each language has separate and distinct cortical centers. Cor-
tical areas for rst acquired language had a larger cortical representation,
whereas those for the secondly acquired languages were localized in more
distinct cortical sites. Subcortical stimulations found tracts for the rst
acquired language in 4 patients, whereas those for the other languages in
3 patients. Subcortical tracts for the rst language had a larger representa-
tion. Three patients experienced a decrease in uency immediately after
surgery, mainly affecting the rst acquired language, which fully recovered
in two patients in two months and partially in one. These ndings support
the existence of language-specic cortical sites and the concept that intra-
operative mapping should be performed for all the languages the patient is
uent for, to maximally preserve functional language integrity. In addition,
confrontation naming test is more accurate than counting for localization
of functional areas.
10. 5-AMINOLEVULINIC ACID-BASED PHOTODYNAMIC
DETECTION OF VARIOUS BRAIN TUMORS
K. Mishima, R. Nishikawa, and M. Matsutani; Saitama Medical School,
Neurosurgery, Saitama-ken, Japan
It has been established that 5-aminolevulinic acid (ALA) induces the
accumulation of uorescent porphyrins in malignant gliomas, a phenom-
enon potentially exploitable to guide tumor resection. However, the use-
fulness of uorescence-guided resections of other brain tumors, especially
low-grade tumors, has not been studied. Here, we examined the value of
ALA-induced uorescence for detecting various brain tumors including
low-grade tumors. Forty-seven patients underwent ALA-induced pro-
toporphyrin uorescence detection. Three hours before the induction of
anesthesia, 1 g 5-ALA/body was administered orally. Intraoperatively, red
porphyrin uorescence was observed with a 455-nm long-pass lter after
excitation with violet-blue (405 nm) light. Fluorescing and nonuorescing
samples taken from the tumor tissues were examined histologically. Bright
red uorescing tumor tissues were observed in 100% (9 of 9) of glioblasto-
mas, 66% (2 of 3) of anaplastic astrocytomas, 100% (3 of 3) of anaplastic
oligodendrogliomas, 0% (0 of 2) of diffuse astrocytoma and oligodendro-
glioma, 100% (3 of 3) of pilocytic astrocytomas, 72% (8 of 11) of menin-
giomas (WHO grade I), 33% (1 of 3) of atypical meningiomas, 62% (5 of 8)
of germ cell tumors, 66% (2 of 3) of malignant lymphoma, and 50% (1 of 2)
of hemangioblastomas. The observations in this study indicate the useful-
ness of 5-ALA-induced tumor uorescence not only for guiding malignant
glioma resection but also for guiding resection of benign tumors such as
pilocytic astrocytoma or meningioma. ALA-mediated uorescence detec-
tion may enhance the completeness of benign tumor removal and increase
the diagnostic accuracy of intraoperative biopsies.
11. VOLUMETRIC EXTENT OF RESECTION AND RESIDUAL
CONTRAST ENHANCEMENT AT INITIAL SURGERY AS
PREDICTORS OF OUTCOME IN ADULT PATIENTS WITH
HEMISPHERIC ANAPLASTIC ASTROCYTOMA
G.E. Keles, E.F. Chang, K.R. Lamborn, T. Tihan, C.-J. Chang, S.M.
Chang, and M.S. Berger; University of California San Francisco,
Department of Neurological Surgery, San Francisco, California, USA.
The available literature evaluating the role of extent of resection for
anaplastic astrocytomas, as a distinct histological group, is limited, and
assessment of residual disease has not been quantitative. To investigate the
prognostic signicance of volumetrically assessed extent of resection on
time to tumor progression (TTP), overall survival (OS), and tumor recur-
rence patterns, we retrospectively analyzed preoperative and postoperative
tumor volumes on 102 adult patients from the time of the initial resection
for a hemispheric anaplastic astrocytoma. Histological diagnosis of ana-
plastic astrocytoma was conrmed based on pathology re-review for all
patients using the current World Health Organization criteria. Patients with
recurrent anaplastic astrocytoma were not included in this study. Quan-
tication of tumor volumes was based on a previously described method
involving computerized image analysis of magnetic resonance imaging
scans. Volumetric analysis was conducted on contrast-enhancing tumor
volumes on T1-weighted MR images for 67 patients who had contrast-
enhancing tumors, in addition to measurements of T2 hyperintensity for all
102 patients. The variables analyzed included age, Karnofsky Performance
Status (KPS), preoperative tumor volume (T1 enhancement and T2 hyperin-
tensity), percent of resection (POR), and volume of residual disease (VRD)
(T1 enhancement and T2 hyperintensity). All patients had postoperative
radiotherapy, and 94% (96/102) of the patients received chemotherapy.
Presence or absence of preresection enhancement, actual volume of this
enhancement, and the percentage of preoperative enhancement as it relates
to the total T2 tumor volume did not have a statistically signicant impact
on TTP or OS. In addition to age, VRD measured on T2-weighted MR
scans was the most signicant predictor of TTP. (Unlike low-grade gliomas,
there was no statistically signicant relationship between extent of resec-
tion, i.e., POR and VRD, and histology at the time of recurrence, i.e., grade
3 vs. grade 4.) This retrospective analysis of a histologically uniform group
of hemispheric anaplastic astrocytomas treated in the MR era suggests that
residual tumor volumes, as documented on postoperative imaging studies,
may be a prognostic factor for time to progression and survival for this
patient population.
12. POST- OPERATIVE OUTCOME OF ANTERIOR SKULL
BASE MENINGIOMAS
M. Kirsch, A. Knppel, and G. Schackert; Carl Gustav Carus University
Hospital, Neurosurgery, Dresden, Germany
The purpose of this retrospective study was to analyze the outcome and
recurrence of anterior skull base meningiomas. A total of 571 meningiomas
were operated upon at the Neurosurgical Department, Technical University
of Dresden, between January 1994 and December 2002. Of these 571 menin-
giomas, 151 were located within the anterior skull base, namely, the fron-
tal base, sphenoid wing, tuberculum sellae, cavernous sinus, and olfactory
groove. One hundred forty-one patients were amendable for follow-up includ-
ing regular ambulatory visits and a questionnaire. The median follow-up
including MR imaging was 2.5 years (0-7.6 years). The median age was 62.5
years, and the male:female ratio was 1:2.5. The patients most commonly
presented with visual decits (37%), headaches (25%), dizziness (16%),
seizures (12%), or symptoms of organic psychosis (11%). Median tumor
volume was 9.7 cm
3
, mean volume was 26.1 35.6 cm
3
. Most frequent his-
tology subtypes were meningothelial (67%) and transitional (18%) menin-
giomas, whereas WHO grade II meningiomas appeared only twice (1.4%).
Seizures were most frequently associated with lateral sphenoid wing and
frontal base tumors, whereas visual acuity changes, dizziness, and double
vision were associated with medial sphenoid wing, olfactory groove, and
tuberculum sellae tumors. Tumor size correlated with appearance of symp-
toms. Intraoperative radicality was in 16% Simpson grade 1, 59% grade 2,
13% grade 3, and 15% grade 4. Biopsies (grade 5) were not done. Operative
radicality was associated with location, with medial sphenoid wing and
cavernous sinus meningiomas being the most difcult to resect completely.
Pre-operative angiography was completed in 30 patients, of which 12 were
embolized. Upon initial post-operative MR imaging, residual or recurrent
tumor was diagnosed in 33% of cases, which was corrected on repeated
follow-up imaging to 12.7%. Simpson grading correlated and angioma-
tous or brous histological subtype correlated with frequency of recurrence.
Glasgow outcome scale rating demonstrated that ve patients (3.6%) died
within six months post-op (grade 1), one patient remained in grade 2, and
80.7% recovered completely and improved to grade 5. Simpson grade 1 and
2 resected tumors were more likely to show a good outcome (GOS 5 in 97%
and 82%, respectively), compared to incompletely resected tumors. Age but
not tumor volume or gender correlated with outcome. Of pre-operatively
full-time-employed patients, 64% were able to return to their previous
workplace, 27% had to start a gradual re-integration program, and 9%
were not able to return to work. Anterior skull base meningiomas can be
resected dependent on their location and involvement of other structures,
such as vessels and cranial nerves. The most important predictors of post-
operative outcome were Simpson grade, age, and location. Repeated MR is
required for reliable assessment of recurrent tumor growth.
13. TAILORING OF ANTI ANGIOGENIC THERAPIES TO
TUMOR STAGE IMPROVES THERAPEUTIC EFFICACY IN
MOUSE MODELS OF HUMAN GLIOMAS
C. Giussani,
1
F. Acerbi,
1
S. Sangiorgi,
2
F. Colleoni,
1
G. Carrabba,
1

V. Lucini,
1
M. Pannacci,
1
G. Tomei,
2
S.M. Gaini,
1
A. Bikfalvi,
3

and L. Bello
1
;
1
University of Milano, Neurosurgery, Department of
Neurological Sciences, Milano, Italy;
2
Neurosurgery, University of
Insubria, Varese, Italy;
3
INSERM Unit EPI 0113, University of Bordeaux I,
Talence, France
Systemic administration of inhibitors of angiogenesis, migration,
and proliferation successfully inhibits the growth of experimental malig-
nant gliomas. Nevertheless, after some time, tumor escapes treatment. In
this work we investigated the role of changes in tumor vasculature dur-
ing glioma development on the signicance of the therapeutic effect. We
initially studied the changes in tumor vasculature occurring in the well-
established and surgical resection glioma models in nude mice. Brains from
animals sacriced at various time points from tumor cell injection or tumor
removal were studied by immunouorescence, immunohistochemical, and
vessel casting techniques. Early tumors were composed of irregular highly
angiogenic vessels, uncovered from pericytes. Tumor vasculature in late
tumors showed a complex regional heterogeneity, with highly angiogenic
areas and areas composed of more regular vessels covered by pericytes.
We then investigated the effect of different inhibitors on various stage of
vasculature development. Inhibitors that exhibit various activities on tumor
or endothelial cells and that act by different mechanisms were adminis-
tered to early, mean, and late tumors in both glioma models. Efcacy was
evaluated by survival in long-term experiments and by evaluating changes
in tumor vasculature by immunouorescence, immunohistochemical, and
vessel casting techniques. PF-4/CTF, a pure anti angiogenic drug that acts
mainly by inhibiting FGF, was more effective on early tumors; PF-4/DLR,
which inhibits FGF and VEGF and acts on both tumor and endothelial cells,
was effective on both early and late tumors. A low-dose chemotherapy regi-
men based on carboplatin and etoposide was more active on late tumors.
Based on these ndings, we designed a scheme of treatment characterized
by the sequential administration of these agents, alone or in combination.
PF-4/CTF followed by PF-4/DLR, initially alone and then in combination
with low-dose chemotherapy, was administered to early tumors. A combi-
nation of PF-4/DLR and PF-4/CTF followed by PF-4/DLR and low-dose
chemotherapy was given to late tumors. Both schemes afforded a prolonged
inhibition of glioma growth, which was more effective than the single
administration of each inhibitor alone or in combination. Taken together,
these ndings indicate that tailoring the scheme of administration to the
stage of vasculature development optimizes efcacy and further postpones
the appearance of tumor escape.
14. VEGF COOPERATES WITH SECRETED PROTEIN
ACIDIC AND RICH IN CYSTEINE (SPARC) TO ACTIVATE
INTRACELLULAR GLIOMA SURVIVAL PATHWAYS
Q. Shi,
1
S. Bao,
1
D. Bigner,
2
and J. Rich; Departments of
1
Surgery,
2
Pathology, and
3
Medicine, Duke University Medical Center, Durham,
North Carolina, USA
Secreted protein acidic, rich in cysteine (SPARC) is an extracellular
matrix protein expressed in many advanced cancers, including malignant
gliomas. We and others have previously shown that human glioma cell lines
engineered to overexpress SPARC adopt an invasive phenotype. We now
show that SPARC expression increases cell survival under stress initiated by
serum withdrawal through a decrease in apoptosis. Phosphatidylinositol 3-
OH kinase (PI3K)/AKT is a potent pro-survival pathway that contributes to
the malignancy of gliomas. Cells expressing SPARC display increased AKT
activation with decreased caspase 3/7 activity. Exogenous SPARC rapidly
induces AKT phosphorylation, an effect that is blocked by a neutralizing
SPARC antibody. Further, AKT activation is essential for the anti-apoptotic
effects of SPARC as the decreased apoptosis and caspase activity associ-
ated with SPARC expression can be blocked with dominant-negative AKT
or a specic AKT inhibitor. As tumor cells face stressful microenviron-
ments particularly during the process of invasion, these results suggest that
SPARC functions, in part, to promote tumor progression by enabling tumor
cells to survive under stressful conditions. We have now probed the signal-
ing events upstream from AKT activation by SPARC. SPARC binds and
regulates growth factor presentation to cells. We examined the impact of
SPARC and several growth factors on activation of intracellular pathways,
including AKT. While IGF1 induced AKT phosphorylation, the addition
of SPARC did not alter this activity. In contrast, VEGF and SPARC each
modestly increased AKT phosphorylation the combination more signi-
cantly induced AKT phosphorylation. Additionally, focal adhesion kinase
(FAK) appears upstream of AKT as both SPARC alone and in combina-
tion with VEGF induced FAK phosphorylation before AKT phosphoryla-
tion. Dominant negative FAK (FRNK) induced cell death suggesting like
AKT in these cells FAK is essential to cell survival. In summary, we have
now directly linked SPARC to essential tumor processes including inva-
sion, survival, and angiogenesis. SPARC warrants further investigation as
a contributor to glioma malignancy and as a potential therapeutic target.
(J.N.R. is a Damon Runyon-Lilly Clinical Investigator and a Sidney Kim-
mel Cancer Foundation Scholar. This work was also supported by NIH
grant NS047409 to J.N.R.).
15. INHIBITION OF INTRACEREBRAL GLIOBLASTOMA
GROWTH BY TREATMENT WITH A NOVEL ONE-ARMED
ANTI-MET ANTIBODY
T. Martens,
1
N.O. Schmidt,
1
C. Eckerich,
1
R. Fillbrandt,
1
R. Schwall,
2

M. Westphal,
1
and K. Lamszus
1
;
1
University Hospital Hamburg-
Eppendorf, Department of Neurosurgery, Hamburg, Germany;
2
Genentech, Inc., South San Francisco, California, USA
The proto-oncogene encoded tyrosine kinase receptor MET and its
ligand scatter factor/hepatocyte growth factor (SF/ HGF) are strongly
upregulated in malignant gliomas. The SF/HGF-MET system is impor-
tant for glioma cell migration, invasion, proliferation, and angiogenesis.
We used a novel single-chain anti-MET antibody to inhibit glioblastoma
growth in an orthotopic model. U87 glioblastoma cells were xenografted
into the brains of nude mice. On day 1 or day 7 after tumor cell injection,
osmotic minipumps with intratumoral catheters were implanted. The one-
armed anti-MET antibody (40 mg/day) was infused intratumorally until
3 weeks after tumor cell injection. Tumor size, proliferation, apoptosis,
microvessel density, and expression of extracellular matrix (ECM) mol-
ecules were analyzed by immunohistochemistry. cDNA arrays were per-
formed to determine the effect of the anti-MET antibody on the expres-
sion of invasion-related genes in vitro. Functional effects of the anti-MET
antibody were analyzed in vitro. The effects of the anti-MET treatment on
tumor size and morphology were very similar, regardless whether treatment
was initiated on day 1 or day 7. Tumor volumes were reduced by 95% (P
0.001) in animals treated with the anti-MET antibody compared with
controls. Tumor cell proliferation was reduced by 75% (P 0.001) in
treated tumors; microvessel density was reduced by 90% (P 0.001);
apoptosis was increased by 60% (P 0.05). Interestingly, the tumor cell
density was 2-fold higher in controls than in treated tumors, in which a
striking increase in ECM deposition between tumor cells was apparent.
Immunohistochemically, strong increases in staining intensities for laminin,
bronectin, and tenascin were found in tumors treated with the anti-MET
antibody. cDNA arrays revealed downregulation of uPA, tPA, MMP7,
MMP15, and MMP16 and upregulation of PAI-1 in U87 cells treated with
the anti-MET antibody, which may explain the increase in ECM proteins in
vivo. Proliferation and migration of U87 cells in vitro were inhibited by the
anti-MET antibody. Local treatment with the one-armed anti-MET anti-
body can inhibit intracerebral glioblastoma growth almost completely. The
responsible mechanisms appear to include anti-proliferative, anti-angio-
genic, anti-migratory, and pro-apoptotic effects as well as enhancement of
ECM deposition, presumably caused by decreased matrix degradation.
16. ANGIOPOIETIN-2 INDUCES GLIOMA CELL INVASION
BY STIMULATING MMP-2, MT1-MMP AND LN 5 GAMMA 2
EXPRESSION THROUGH TIE2-INDEPENDENT PATHWAYS
B. Hu,
1
P. Guo,
2
M.J. Jarzynka,
2
R. Nishikawa,
3
T. Hirose,
4
and S.-Y.
Cheng
2
; Cancer Institute and Departments of
1
Medicine and
2
Pathology,
University of Pittsburgh, Pittsburgh, Pennsylvania, USA; Departments of
3
Neurosurgery and
4
Pathology, Saitama Medical School, Saitama, Japan
A hallmark of malignant human gliomas is their ability to diffusely
invade into surrounding brain tissues. We have previously reported that
angiopoietin-2 (Ang2), a known angiogenic factor, induces glioma cell inva-
sion through the activation of matrix metalloprotease-2 (MMP-2). In this
study, we analyzed 57 human glioma biopsies (WHO grade I to IV) display-
ing a distinct invasive edge and 39 glioma specimens that only contain the
central region of the tumors and found that Ang2, MMP-2, MT1-MMP,
and LN 5 gamma 2 were co-overexpressed in the invasive areas, but not
in the central regions of the glioma tissues. Statistical analyses revealed
a signicant link between the preferential expression of these molecules
and invasiveness. Western blot analyses of total protein extracted from the
microdissected primary glioma specimens showed an upregulation and
activation of MT1-MMP and LN 5 gamma 2 at the invasive edge of the
tumors versus the tumor center, supporting this observation. Analysis of
engineered U87MG glioma xenografts which express Ang2, revealed an
increased expression of MT1-MMP and LN 5 gamma 2 in actively invad-
ing glioma cells, along with MMP-2 upregulation. Stimulation of glioma
cells by overexpressing Ang2 or exposure to exogenous Ang2 promoted the
expression and activation of MMP-2, MT1-MMP, and LN 5 gamma 2. Fur-
thermore, Ang2 directly binds to beta 1 integrin in Tie2-decient U87MG
cells inducing the activation of FAK, p130Cas, ERK1/2, and JNK. Ang2-
stimulated MMP-2 expression and secretion was attenuated by a functional
neutralizing anti-beta 1 antibody, by an ERK1/2 inhibitor, and by a JNK
inhibitor. Stable expression of a specic negative regulator of FAK, FAK
related non-kinase (FRNK) but mutant FRNK-S1034, inhibited Ang2-
stimulated phosphorylation of FAK and p130Cas, blocked the activation
of ERK and JNK, and decreased Ang2-stimulated MMP-2 expression and
secretion. Inhibition of beta 1 integrin, FAK, p130Cas, ERK1/2, and JNK
also attenuated Ang2-stimulated glioma cell invasion. Lastly, expression of
FRNK, but not FRNK-S1034, by glioma xenografts in the brain derived
from U87MG/Ang2 cells suppressed Ang2-induced glioma cell inltration
and MMP-2 expression. These data suggest that upregulation of Ang2,
MMP-2, MT1-MMP and LN 5 gamma 2 is associated with glioma inva-
siveness and demonstrate a mechanism whereby binding of Ang2 to glioma
cells regulates MMP-2 expression and secretion through the integrin and
FAK signaling pathways.
17. PROMOTION OF MALIGNANT GLIAL CELL POLARITY
AND INVASION BY DRR-1
R. Waldkircher de Oliveira, K. Petrecca, A. Angers-Loustau, M. Seyed-
Sadr, and R. Del Maestro; Montreal Neurological Institute, Neurology/
Neurosurgery, Montreal, Canada
Invasion is a major factor responsible for the failure of brain tumor
treatment. There are currently no chemotherapeutics directed at controlling
the inltration of malignant glial cells into normal brain. In order to iden-
tify invasion-associated genes we designed an unbiased, genome-wide func-
tional screen based on tumor cell invasion. A normal adult human brain
cDNA library was stably infected into a glioma cell line using retroviral
transduction. Tumor spheroids generated from these cells were implanted
into a 3D collagen matrix. Hyperinvasive cells were isolated from the matrix
and cDNA inserts identied. Downregulated in renal cell carcinoma (Drr1)
was identied as a potent mediator of glial cell hyperinvasion in this screen.
Time-lapse video microscopy reveals that Drr1 overexpression promotes
invasion, whereas RNAi-mediated expression reduction inhibits invasion in
a 3D tumor model. Importantly, human glioma sampling reveals that Drr1
is highly expressed in invasive gliomas, whereas expression is not detectable
in noninvasive gliomas. Further, we have identied Drr1 as a novel actin-
microtubule polylinker that promotes invasion by enhancing cell polarity
using a pericentrin localization assay. These ndings uncover a novel glioma
invasion gene and its mechanism of action, which provides an explanation
for the highly invasive nature of Drr1-expressing human gliomas.
18. PHARMACOLOGIC INHIBITION OF MTOR
ACTIVITY ATTENUATES GLIOMA CELLS INVASION BY
DOWNREGULATING NEUROPILIN-1 EXPRESSION
T.J. Liu,
1
T. LaFortune,
1
J. de Groot,
1
H. Lane,
2
and W. Yung
1
;
1
The
University of Texas, M.D. Anderson Cancer Center, Neuro-Oncology,
Houston, Texas, USA;
2
Novartis Pharma AG, Basel, Switzerland
The mammalian target of rapamycin mTOR controls a spectrum of cel-
lular events such as initiation of translation, ribosome biogenesis, and cell
growth and proliferation. Pharmacologic inhibition of mTOR activity by
rapamycin has been shown to elicit antitumor activity possibly through G
1

cell cycle arrest and inhibition of VEGF expression. Currently, an analogue
of rapamycin, RAD001, is being used in clinical trial for different cancers
including gliomas. Glioblastoma multiforme is a malignant tumor that is
extremely refractory to therapy because of rapid growth and local invasive
potential of these tumors. In this study, we sought to examine the effect
of antagonizing mTOR activity by RAD001 on glioma tumor invasion in
vitro. Four different glioma cells were treated with RAD001 for 72 h before
harvesting for Western blotting, RT-PCR, microarray, and in vitro Matri-
gel gel invasion analyses. Glioma cells were stably transfected with NRP-1
expression and control vector followed by G418 selection. The resulting
G418-resistant cells were examined for their NRP-1 expression and in vitro
invasion propensity. Glioma cell lines treated with RAD001 resulted in a
reduction of mTOR downstream target genes expression such as S6K1 and
the eukaryotic initiation factor 4E-binding protein 1. Inhibition of mTOR
activity with RAD001 in tumor cells also leads to G
1
cell cycle arrest and
reduction in VEGF secretion consistent with previously documented stud-
ies. Importantly, invasion propensity of tumor cells is greatly inhibited by
RAD001 as assessed in an in vitro Matrigel invasion assay. Interestingly,
RAD001 treatment does not affect either the expression or the activity of
MMP2 in tumor cells. Using Affymetric microarray analyses we discovered
the expression of neuropilin-1 (NRP-1) is decreased by RAD001. NRP-1,
initially found to be involved in axon growth during neuronal development,
is expressed in both tumor cells and endothelial cells. It has been shown
that NRP-1 is a co-receptor for VEGF
165
and controls cell motility. Semi-
quantitative PCR further conrmed the microarray results, which suggests
that RAD001 affects NRP-1 mRNA expression. Exogenous NRP-1 expres-
sion signicantly increases glioma cells invasion and promotes anchorage-
independent growth. Our results demonstrate that anticancer activity of
RAD001 may be a combination of growth arrest, antiangiogenesis, and
anti-invasion effects. Furthermore, the potential anti-invasion activity of
RAD001 is likely through controlling cell motility by inhibiting NRP-1
expression.
19. POLYMORPHISMS IN DNA REPAIR GENES AND
SUSCEPTIBILITY TO PRIMARY INTRACRANIAL BRAIN
GLIOMAS
M. Butler,
1
A. Ruder,
1
T. Carreon,
1
M. Waters,
1
M. Yeager,
2
R. Welch,
2

S. Chanock,
2
and P. Schulte
1
;
1
National Institute for Occupational Safety
and Health, Cincinnati, Ohio;
2
National Cancer Institute, Gaithersburg,
Maryland; USA
Enzymes in base excision (BER), nucleotide excision (NER), double strand
break/recombination (DSB/RR), mismatch (MMR), and direct-damage
DNA repair pathways are important in the repair of diverse types of DNA
damage. Polymorphisms in many of the genes encoding these enzymes
have been identied as risk factors for environmentally and occupation-
ally caused cancers. We evaluated the associations of polymorphisms in
BER (ADPRT V762A, APEX D148E, MUTYH Ex18ACG/T, OGG1
S326C, POLB IVS11-235AG, XRCC1 R399Q, R280H, R194W, LIG1
Ex2-24CT, PCNA IVS1-124CT), NER (ERCC2 D312N, K751Q,
ERCC4 R415Q, ERCC5 H1104D, RAD23B A249V, LIG1 Ex2-24C
T, PCNA IVS1-124CT), DSB/RR (NBS1 Q185E, RAD52 Y415stop,
XRCC2 R186H, XRCC3 T241M, XRCC4 N298S), MMR (MLH1 I219V,
MSH2 G322D), and direct-damage repair (MGMT I143V, R178K, L84F) as
risk factors for primary intracranial gliomas in the Upper Midwest Health
Study, a population-based case-control study including rural residents of
four states with high glioma incidence. Glioma cases (N 798) were identi-
ed from hospitals, private physicians, and registries. Control participants
(N 1175) were stratied samples of licensed drivers and HCFA enrollees.
Questionnaires elicited occupational and environmental exposures. DNA
was obtained from 451 controls with no self-reported cancer and from 316
cases. TaqMan and MGB Eclipse methodology were used to characterize
genotypes. In unadjusted analyses, a polymorphism in ADPRT (V/V 67%
of controls, 75% of cases, odds ratio [OR] 1.48, 95% condence interval
[CI] 1.072.04) had a statistically signicant association with glioma, and
polymorphisms in three other genes showed associations with glioma of
borderline statistical signicance: (1) RAD23B A/V V/V, 32% of con-
trols, 38% of cases, OR 1.32, CI 0.971.78; (2) ERCC5 H/H, 61% of
controls, 68% of cases, OR 1.33, CI 0.981.78; and (3) XRCC4 N/N 74%
of controls, 80% of cases, OR 1.37, CI 0.971.94. For each DNA repair
pathway, multivariate logistic analyses included all polymorphisms in the
pathway plus ever/never living on a farm and ever/never smoking, as sur-
rogates for occupational and environmental exposure. Adjusting for these
factors did not change odds ratios substantially. Our results should be con-
rmed in additional glioma case-control studies. Future analyses of our
data will include assessing the risk of DNA repair polymorphisms under
specic exposure conditions, such as exposures to pesticides, solvents, and
UV light.
20. A GLTSCR1 HAPLOTYPE IS ASSOCIATED WITH
OLIGODENDROGLIOMA DEVELOPMENT
R.B. Jenkins,
1
T. Kollmeyer,
1
K. Buckner,
1
W. Bamlet,
2
K.V. Ballman,
3

and P. Yang
2
;
1
Mayo Clinic, Division of Laboratory Genetics;
2
Mayo
Clinic, Department of Health Sciences Research;
3
Mayo Clinic, Cancer
Statistics Unit, Rochester, Minnesota, USA
Deletions of 19q have been associated with gliomas, especially oligo-
dendrogliomas. In addition, oligodendrogliomas with 19q deletion have a
better survival. We have previously described a 150-kb minimal deletion
region in gliomas that maps to 19q13.33 that contains three novel candi-
date genes (GLTSCR1, EHD2, and GLTSCR2). Polymorphisms in loci
near this deletion region (ERCC1, ERCC2, RAI, ASE-1, and D19S246)
have been associated with basal cell, breast and lung carcinoma, and mixed
oligoastrocytomas. A polymorphism within GLTSCR1 has recently been
associated with prostate cancer aggressiveness. We have recently shown
that a polymorphism in GLTSCR1 (SNP rs1035938) is associated with
oligodendroglioma development. We have now evaluated ve additional
SNPs within GLTSCR1 using the same cohort of glioma cases and general
controls. One of the polymorphisms (Novel 4 A to G at position 304
from the putative transcription start site) is a novel SNP discovered during
GLTSCR1 mutation screening studies of sporadic glioma specimens. Of
these ve SNPs, two (Novel 4 and rs1005911) were found to have bor-
derline association with oligodendroglioma development by allele-based
analysis (P for both 0.052). However, the prevalence of the at-risk allele
was signicantly increased in patients whose oligodendroglioma have 19q
deletion (P for Novel 4 0.025; P for rs1005911 0.014). By genotyping
10 CEPH families we were able to determine the most prevalent haplotypes
for the six total GLTSCR1 SNPs we tested. We then assessed the prevalence
of these haplotypes in the cases with glioma and the normal controls. One
haplotype (haplotype 1, ACTCGG) was more prevalent in gliomas than
in controls (27.5% vs. 20.4%, P 0.067). The prevalence increased to
36.7% in gliomas with 19q deletion (P vs. controls 0.009; P vs. gliomas
without 19q deletion 0.02). The increased prevalence of this haplotype in
gliomas was primarily due to its prevalence in oligodendrogliomas (34%, P
vs. controls 0.010) and among the oligodendrogliomas, those with 19q
deletion (45%, P vs. controls 0.001; P vs. oligodendrogliomas without
19q deletion 0.002). Interestingly, this haplotype is retained in 8 of 10
oligodendrogliomas with 19q deletion. Even though it had a low overall
prevalence, another haplotype (ACCCGG) was signicantly more prevalent
in the controls than the cases (2% versus 0%; P 0.035). The high-risk and
low-risk haplotypes only differ by the presence of the GLTSCR1-exon-1 T
allele (rs1035938). These preliminary data strongly suggest that a polymor-
phism (mutation) is in linkage disequilibrium with the GLTSCR1 ACTCGG
haplotype and is associated with oligodendroglioma development, espe-
cially those with 19q deletion.
21. NON-ENZYME-INDUCING ANTIEPILEPTIC DRUG
USE IN BRAIN TUMOR PATIENTS: EXPERIENCE WITH
LEVETIRACETAM
G.J. Stevens, G.H. Barnett, M.A. Vogelbaum, and D.M. Peereboom;
Cleveland Clinic Foundation, Brain Tumor Institute, Cleveland, Ohio,
USA
It has been estimated that 20% to 40% of brain tumor patients experi-
ence a seizure by the time of diagnosis, accounting for 60,000 patients
annually. The hepatic enzyme-inducing medications such as phenytoin and
carbamazepine have been the drugs of choice for treatment. There may
be advantages to using non-enzyme-inducing drugs such as levetiracetam
(LEV) to help decrease medication interactions. A retrospective analysis
was completed using our brain tumor database that identied all brain
tumor patients treated over the past three years with LEV. Patients were
evaluated for tumor type, seizure type and frequency, and medication side
effects. At the time of the abstract, 278 brain tumor patients were identied.
The breakdown included 91 glioblastomas, 24 anaplastic astrocytomas, 15
anaplastic oligodendrogliomas, 13 mixed anaplastic gliomas, 24 low-grade
astrocytomas, 46 low-grade oligodendrogliomas, 15 mixed low-grade glio-
mas, 19 meningiomas, 18 metastases, and 13 tumors dened as other. Of
the 278 patients, 10 (3.5%) stopped or dose reduced the LEV for behavioral
reasons. LEV produced a 50% reduction in seizure activity in 60% of
patients, and 70% of patients were maintained on LEV as monotherapy.
Brain tumor patients are often on multiple medications, which can increase
the possibility of drug interactions. For the past 3 years we have been using
the non-hepatic enzyme-inducing drug LEV for brain tumor patients requir-
ing seizure medication. LEV appears well tolerated and an effective drug to
control seizures in brain tumor patients. A detailed breakdown of LEV use
in brain tumor patients will be presented.
22. PROSPECTIVE QUALITY OF LIFE ASSESSMENT USING
EORTC QLQ 30 AND BRAIN CANCER MODULE (BN 20) IN
257 ADULT PATIENTS WITH PRIMARY BRAIN TUMORS
SEEN CONSECUTIVELY IN A TYPICAL NEUROONCOLOGY
CLINIC
R. Jalali,
1
A. Budrukkar,
1
R. Kamble,
2
R. Sarin,
1
and S. Parab
2
;
1
Tata
Memorial Hospital, 113, Radiation Oncology, and
2
Brain Tumor
Foundation, Tata Memorial Hospital, Mumbai, India
A majority of quality of life (QOL) data for patients with brain tumors
is available as a part of some other study, and there is a relative lack of
information in patients seen routinely in clinical practice. The purpose of
this study was to evaluate QOL in patients with primary brain tumors as
seen in a typical neurooncology clinic, in our setup using validated local
language questionnaires. Two hundred and fty-seven adult patients pre-
senting consecutively in our neurooncology clinic from 1 January 2003 to
31 December 2003 were prospectively accrued in the study. A majority of
the patients had some sort of surgical intervention before the accrual. All
patients underwent a detailed neurological assessment, evaluation of QOL
using EORTC questionnaire (QLQ-30), specic Brain Cancer module (BN
20), and daily activities by modied Barthels index. Assessments were done
before starting treatment (typically radiation therapy [RT]) or chemother-
apy [CT] in some patients), at completion of RT/CT, and at each follow-up
(at least two). The questionnaires were administered in English, Hindi, and
Marathi according to the patients needs using EORTC-ratied versions of
Hindi and Marathi (local Indian languages). Mean post-treatment follow-
up QOL was compared with the pre RT values. Initial evaluation was done
for patients accrued in the rst 6 months (N 137), including 48% below
the age of 40 years, 45% between 41 and 60 years, and 6% above 60 years
with male-to-female ratio of 2.5:1. Eighty-ve percent of the patients had
at least primary education, and KPS was above 90 in 68% of the patients.
Ninety-seven patients received RT up front and form the analyzed patient
population. Sixty-one percent of the patients completed the questionnaire
by themselves, 16% required assistance because of poor neurological condi-
tion, 11.5% because of illiteracy, and 11.5% because of other causes. At the
end of RT, there was a statistically signicant improvement in the overall
global QOL score, the benet of which continued at 3 months. Signicant
improvement was seen in functional scores of physical (P 0.000) and role
domain (P 0.007), while the difference in the emotional, cognition, and
social scores were not signicant. There was deterioration in the symptom
scale with respect to nausea and vomiting (P 0.04). In the BN 20 module
there was a signicant deterioration in scores of hair loss (P 0.001) and
local itching (P 0.01). QOL assessment using EORTC QOL 30 and BN20
in validated local language is simple to administer, is generally well under-
stood, and can be used even in routine patients in a busy neurooncology
clinic. Patients receiving RT as an up-front modality showed a signicant
improvement in global, physical, and role functioning and transient worsen-
ing in scores of hair loss and itching of skin. An update of all 257 patients
at an extended follow up will be presented in the meeting.
23. HUMAN TELOMERASE GENETIC VARIATION
PREDICTS SURVIVAL OF PATIENTS WITH GLIOBLASTOMA
MULTIFORME
L. Wang, R. El-Zein, K. Aldape, M. Gilbert, Q. Wei, and M. Bondy;
M.D. Anderson Cancer Center, Neuro-Oncology, Houston, Texas, USA
Glioblastoma multiforme (GBM) is the most common glioma with the
poorest survival. Although extent of surgery or a combination of radiother-
apy or chemotherapy may improve the outcome, it is important to identify
and evaluate biomarkers that might be useful for screening patients and
possibly modifying treatment. Level of human telomerase (hTERT) mRNA
or protein expression has been comprehensively evaluated in most primary
tumors for therapeutic purpose. A functional variant of hTERT MNS16A-
short tandem repeats (S allele) is known to be associated with higher expres-
sion levels of hTERT mRNA compared with the MNS16A-long (L) allele. In
this study, we investigated whether the hTERT MNS16A variant genotype
predicted survival benet for 362 patients with GBM. All patients were
surgically resected, with some having more complete resection, received
radiotherapy, chemotherapy, or a combination. We found a signicantly
different survival outcome among patients with different hTERT MNS16A
genotypes, with the median survival of 24.7 months (95% CI 14.729.7)
for the SS-genotype, 14.0 months (95%CI13.215.4) for the SL-genotype,
and 13.1 months (95% CI 12.215.7) for the LL-genotype (P 0.0131
by Log-Rank testing). These data suggest a dominant effect of the L allele.
Compared with the SS-genotype, the hazard ratio (HR) of the combined SL
and LL-genotype was 1.83 (95% CI 1.182.83, P 0.007) after adjust-
ment for age, sex, and extent of surgery, and 2.00 (95% CI 1.293.09, P
0.002) after adjustment for age, sex, combined surgery, chemotherapy,
and radiotherapy. On the basis of these observations, we conclude that the
functional MNS16A-genotype of hTERT may modify survival of GBM
and serve as a potential biomarker to assess treatment outcomes. However,
larger studies are needed to verify our ndings.
24. VALIDATION OF BLINDED EVENTS REVIEW
COMMITTEE (ERC) DETERMINED TIME TO NEUROLOGIC
PROGRESSION (TTNP) DEMONSTRATES CORRELATION
WITH SURVIVAL, RADIOLOGIC PROGRESSION, AND
FUNCTIONAL INDEPENDENCE END POINTS
M.F. Renschler,
1
J.A. Smith,
1
W.R. Shapiro,
2
R.A. Patchell,
3
M.J.
Glantz,
4
L.D. Recht,
5
M.P. Mehta,
6
and S.C. Phan
1
;
1
Pharmacyclics,
Sunnyvale, California;
2
Barrow Neurological Institute, Phoenix,
Arizona;
3
University of Kentucky, Lexington, Kentucky;
4
University of
Massachusetts, Worcester, Massachusetts;
5
Stanford University, Palo
Alto, California;
6
University of Wisconsin, Madison, Wisconsin; USA
Survival is a standard end point for brain metastasis trials, but it inad-
equately measures treatment benet because of competing risks for death
from systemic disease, and it does not account for neurologic function,
an important quality of life consideration. There is no standardized and
validated tool to measure TTNP. The purpose of this study was to test the
validity of ERC-determined TTNP. In a prospective, randomized phase 3
trial of whole brain radiation motexan gadolinium for patients (pts)
with brain metastases, 401 pts underwent standardized neurologic exam,
neurocognitive tests (NCT), evaluation of symptoms, functional indepen-
dence (Barthel Index), and MRI. Using a prespecied algorithm, the ERC
reviewed blinded clinical data, excluding MRI, to score progression if 2
of 3 functional domains (NCT, neuro exam, neuro symptoms) showed
progression on consecutive visits (Mehta, J. Clin. Oncol. 21, 2529, 2003).
TTNP was compared with survival, MRI progression, and time to loss of
functional independence by using log-rank tests and Kaplan-Meier plots.
Patients with TTNP 1, 2, 3, or 4 months had a shorter median survival
(by 4.4, 3.9, 3, and 2.4 months, respectively), compared with pts who did
not have neurologic progression at those times. The validity of a blinded
ERC-determined TTNP end point was demonstrated by high correlations
with survival, radiologic progression, and loss of functional independence
end points. We conclude that ERC-determined TTNP measures clinical
benet and is sensitive to change.
25. ADVANCES IN DRUG DELIVERY
M. Westphal; Department of Neurosurgery, University Hospital
Hamburg Eppendorf, Germany
Drug therapy for gliomas has been consistently hampered by lack of
specicity, systemic toxicity, lack of penetration, limitations to size of the
molecules, and poor diffusion into normal brain. Therefore, methods have
been developed for local delivery. Intracavitary delivery has been used
mostly in the context of slow-release biodegradable polymers, which has
resulted in high local drug concentration but variable depth of diffusion
into the tissue. Releasing BCNU, this technique has gone through successful
phase 3 trials. Under investigation for the same technique are different com-
pounds, modied release kinetics, higher doses, and drug combinations.
Pericavitary injection of slow-release polymer with 5-FU after resection
of glioblastoma is used as a radiation sensitizer, and a phase 2 study is just
published and phase 3 in preparation. A major advance is the direct, slow
infusion of therapeutic compounds into tissue, either the tumor or the sur-
rounding normal brain. This convection-enhanced delivery (CED) allows
for any substance to be delivered on the other side of the blood-brain bar-
rier. Large and complex molecules can be used, with their distribution prop-
erties depending on their physicochemical characteristics (charge, solubility,
size). Presently, large fusion molecules of ligands for cell surface receptors
with toxins are under evaluation in phase 2 and 3. Being directed at cell
surface molecules expressed only on tumor cells and not on normal brain
cells, this therapy is aimed to be in the crosshairs of compartmental delivery
and a compartmental specicity. Many more molecules can be designed,
depending only on compartmental specicity, especially antibodies, either
naked or coupled to effector molecules. Distribution will be different for
every new compound and will also depend on heterogenous tissue factors
like scars, cysts, and hemorrhages in a tumor but also favorable white mat-
ter tracts and possibly less permissive areas like basal ganglia or areas of
prior injury in normal brain. Further development requires cooperation
with neuroradiologists to study uid movements with dti, which does not
really prove where the compounds go. Also, computer modeling is currently
being explored for its predictive value to create isodistribution curves. In
addition to the large protein-based therapeutics, classical compounds like
taxol are under investigation for CED. Prodrug therapy is another way to
reduce systemic toxicity but requires the specic transduction of glioma
cells with a converting enzyme. This is achieved by viral or liposomal vec-
tors, and a new phase 3 trial based on a TK transducing adenovirus will
start in 2005. Research into the biology of the specialized endothelial cells
of the blood-brain barrier has revealed transporter molecules which when
employed experimentally may afford specic delivery of conjugated thera-
peutics across the blood-brain barrier.
26. ADVANCES IN MOLECULAR DIAGNOSIS
A. Perry; Washington University, Neuropathology, St. Louis, Missouri,
USA
Diffuse gliomas are clinically, pathologically, and genetically heteroge-
neous malignancies. Patient age and histology are the two most powerful
prognostic parameters, though there remains signicant clinical variability,
both in terms of overall survival and response to therapies. Our understand-
ing of glioma tumorigenesis and progression has advanced greatly over the
last 15 years, though few observations have successfully translated into
molecular diagnostic assays applied daily in neuropathology and neuro-
oncology. The most common is chromosome 1p and 19q testing with com-
bined 1p/19q deletions identifying genetically favorable oligodendroglial
tumors with enhanced survival and responsiveness to both alkylating che-
motherapeutic agents and radiation. It is predominantly, but not exclusively,
associated with histologically classic grade II and III oligodendrogliomas
(ODG), where codeletions are found in up to 80% to 90%, suggesting that
this represents an early event. It is rare in pediatric ODGs, which suggests
an alternate tumorigenic pathway in children. Mixed oligoastrocytomas are
diagnostically challenging and are genetically heterogeneous. We recently
found that survival was enhanced in those with 1p/19q codeletion, 19q
deletion alone, or no detectable alterations, whereas it was decreased in
those harboring 9p deletion, 10q deletion, and/or EGFR amplication.
Small interstitial 1p and/or 19q deletions are also seen in some astrocyto-
mas, though the whole arm 1p/19q codeletions are fairly specic to ODGs.
We have not seen them in morphologic mimics, such as DNT, clear cell
ependymoma, central neurocytoma, and small cell glioblastoma. The latter
enters the differential diagnosis most often. However, survival is short and
it is genetically characterized by 10q deletion (95%), EGFR amplication
(70%), and EGFR-vIII expression (50%). We have also seen rare extraven-
tricular neurocytomas with 1p/19q codeletion, though there is evidence
that some oligodendrogliomas undergo neuronal differentiation and these
2 entities are likely related. Lastly, high-throughput technologies, such as
expression proling and array CGH, provide new opportunities to identify
candidate genes and groups of genes that may lead to molecular diagnostics
applications in the future. The ultimate goal will be to provide accurate and
cost-efcient glioma phenotyping that will enable targeted therapies with
the highest likelihood of success for each individual patient.
27. MALIGNANT GLIOMA: ADVANCES IN RADIATION
THERAPY
M. Mehta; University of Wisconsin, Human Oncology, Madison,
Wisconsin, USA
Randomized clinical trials have established a survival benet with adju-
vant radiotherapy for malignant glioma, and indirect evidence supports a
dose-survival relationship, between 0 and 60 Gy. Escalation beyond 60
Gy has been limited primarily by toxicities, but innovative approaches to
achieve this aim have been developed, piloted, and after promising initial
results, tested in phase 3 trials, only to result in negative ndings. The best
examples of these technologies include brachytherapy and radiosurgery, for
both of which, promising phase 2 results were not corroborated in phase
3 trials. New technologies continue to be developed with the ultimate
objective of achieving dose-escalation. Examples include 3-dimensional
radiotherapy delivery techniques, with which doses up to 90 Gy have been
explored (without improved local control), the balloon brachytherapy
device known as Gliasite, intensity modulated radiotherapy (IMRT), etc.
The purpose of IMRT is to produce exquisite shaping of the radiation dose-
distribution to mimic the exact shape of the tumor, with a dramatic avoid-
ance of nearby critical structures. The value and success of this modality is
contingent on the hypothesis that prior dose-escalation efforts have failed
as a consequence of inadequate target denition due to inherent limitations
of current MRI methods. Perhaps with the advent of functional imaging
with MR and PET, a more precise denition of the target might be achieved,
and the irregularly shaped tumor could be accurately and conformally tar-
geted by using IMRT for dose-escalation, ultimately resulting in improved
local control. Other strategies have been used to achieve dose-escalation
focus on molecular targeting. In the simplest of these approaches, a tumor-
specic antigen is targeted with a locally instilled radiolabeled antibody to
achieve a high target dose, and clinical trials evaluating this approach are
under way. Other targeted approaches have attempted to identify molecu-
larly targeted pathways that support proliferation and angiogenesis and
circumvent apoptosis, with the demonstration of inhibitors of these path-
ways as potent radiosensitizers. Proof-of-principle of this concept comes
from a recent phase 3 head and neck cancer trial. The EGFR and PI3 kinase
pathways are two key examples of this, and initial clinical trials targeting
one or both of these have been launched, and some preliminary data are
available, which will be presented. Other radiosensitizers such as MGd and
RSR-13, already in clinical testing, will be described. Historically, neutron
irradiation has been explored as a way of increasing tumor dose-intensity,
by exploiting the preferential dose-localization properties of neutron inter-
action with elements such as boron, which have a high cross-sectional
area of intersection for neutrons. In order for such a strategy to be suc-
cessful, tumor-specic preferential localization of boron needs to occur at
an adequate concentration level. First- and second-generation boronated
compounds have not met these criteria, and newer efforts are focused on
two fronts: (1) developing tumor-targeted boron-containing compounds
by tagging EFGR ligands and (2) evaluating tumor-specic gadolinium-
containing agents, since gadolinium has a superior cross-sectional area of
intersection with neutrons.
29. DEVELOPMENTAL ABNORMALITIES AND
ONCOGENESIS IN THE BRAIN OF A TRANSGENIC E2F1
MOUSE MODEL
M.V. Olson, D.R. Medrano, H. Jiang, M.G. Lemoine, C. Gomez-
Manzano, M.M. Alonso, K. Aldape, R. Price, W.K.A. Yung, and
J. Fueyo; M.D. Anderson Cancer Center, Neuro-Oncology, Houston,
Texas, USA
The E2F family of transcription factors are involved in tumor suppres-
sion and tumor generation. Although the Rb/E2F pathway is deregulated
in most brain tumors, there is no direct evidence of the role of E2F1 in the
generation or maintenance of brain tumors. To address this question, we
generated a transgenic animal model driving expression of E2F1 through
the GFAP promoter to glial cells and neuronal/glial precursors. Histological
analysis of brain tissue revealed expression of the transgene in astrocytes,
ependyma, and the Bergman glial cells of the cerebellum. Importantly,
cells positive for E2F1 were also positive for proliferation markers such as
PCNA and BRDU as well as apoptosis, detected through TUNEL assay.
Overexpression of E2F1 led to the generation of a phenotype character-
ized by numerous neurological defects without a clear pathological frame.
The early onset of these phenotypes suggests a developmental abnormal-
ity. When overexpression of E2F1 was introduced into an E2F4 null back-
ground, a new phenotype, not seen in either background alone, was induced
and characterized by the development of a domed head, hypo- and hyper-
activity, and seizures. MRI examination of these mice showed a dramatic
hydrocephalus. Pathologic examination of the brains uncovered congenital
triventricular hydrocephalus due to E2F1-induced hyperproliferation of the
ependyma in the aqueduct, resulting in the death of the animal by 5 weeks
of age. Parallel studies involving the overexpression of E2F1 and simulta-
neous inactivation of p53 resulted in neurological signs including tremors,
ataxia, seizures, head tilt and paresia of the posterior limbs. Histological
analyses of the brains from 3-month-old transgenic animals uncovered
the production of neoplasms including a highly undifferentiated choroid
plexus carcinoma with papillary and glandular features and an aggressive
embryonal tumor of the cerebellum, expressing the pathologic features of
medulloblastoma. Furthermore, immunohistochemical studies conrmed
proliferation of cells expressing the hE2F1 transgene within the tumor tis-
sue. This study is the rst to provide direct evidence that E2F1 functions as
an oncogene through the induction of brain cancer.
30. FOS-RELATED ANTIGEN 1 (FRA-1) MODULATES
MALIGNANT FEATURES OF GLIOMA CELLS
W. Debinski and D.M. Gibo; Wake Forest University, Brain Tumor
Center of Excellence, Winston-Salem, North Carolina, USA
We uncovered that a c-fos inducible vascular endothelial growth factor
D (VEGF-D) is ubiquitously upregulated in high-grade gliomas (HGG).
However, HGG overexpress a Fos-related antigen 1 (Fra-1) rather than
c-Fos among the activating protein 1 (AP-1) transcription factors. There-
fore, we have examined an effect of ectopic Fra-1 or a knockdown of this
transcription factor in H4 (low Fra-1, non-tumorigenic), U-87 MG (high
Fra-1, highly tumorigenic), and A-172 MG (moderate Fra-1, non-tumori-
genic) malignant glioma cells. We have transfected glioma cells with fra-1
in both sense () and anti-sense (-) orientation. The ectopic Fra-1 evoked
prominent phenotypic changes in all cell lines studied: The cells became
more polar with larger number of elongated processes. This was seen by
standard microscopy and by changes in actin architecture using phalloidin
staining. We noticed that fra-1 siRNA, but not nonsense nucleotides, pro-
duced reversal of the morphological features associated with the ectopic
Fra-1 (H4). The characteristic change in the phenotype seen in cell in vitro
was carried over to tumors grown in vivo (U-87 MG). Of interest, com-
pletely non-tumorigenic H4 cells started to form tumors when transfected
with fra-1 transgene, at an 80% of tumor take. Moreover, the genotype of
H4[fra-1]() cells changed signicantly, since 18 different genes became
overexpressed, at least fourfold vs. controls, with a targeted cDNA micro-
array analysis (1056 genes). Conversely, fra-1(-) altered prominently the
morphology (U-87 and A-172), anchorage-independent growth (U-87 and
A-172), tumorigenic potential (U-87), and the expression of Fra-1 effectors,
such as VEGF-D (U-87 and A-172). For example, fra-1(-) made cells more
rounded, with fewer and shorter cellular processes (U-87 and A-172). Also,
the U-87[fra-1](-) cells lost an ability to grow in agar, while U-87[fra-1]()
cells started to form multiple colonies; similar results were seen in A-172
cells. Furthermore, we found that by day 22, there were 80% U-87[fra-1](-)
tumors formed (8 out of 10) of an average size 20 mm
3
while the size
of U-87[fra-1]() tumors (10 out of 10) was 135 mm
3
. In addition, the
U-87[fra-1](-) tumors were poorly vascularized, and the levels of VEGF-D
in tumor cells were low compared to parental U-87 tumors. Thus, Fra-1
induces profound phenotypic changes in malignant glioma cells with asso-
ciated signicant changes in their genotype. Fra-1 engages mechanisms
that promote tumorigenesis and anchorage-independent growth. Being
that Fra-1 is frequently upregulated and also accumulates to high levels in
response to AP-1 activation in malignant glioma cells, this transcription
factor may likely play an important role in the maintenance or progression
of malignant gliomas and potentially represents a new target for therapeutic
interventions.
31. HUMAN BRAIN SLICES AS CONTROL ASSAY FOR
TUMOR-TARGETED THERAPY IN VITRO
R.P. Weenink,
3
C.M.F. Dirven,
2
W. van Houdt,
2
D.P. Noske,
2
S. Idema,
2

M.L.M. Lamfers,
2
V.W. van Beusechem,
1
and R.W.H. Verwer
3
;
1
VU
University Medical Center, Gene Therapy;
2
VU University Medical
Center, Neurosurgery;
3
Netherlands Institute for Brain Research,
Amsterdam, The Netherlands
To develop novel tumor-targeted anticancer drugs, such as oncolytic
adenoviruses, early in vitro testing of efcacy is a prerequisite. True selec-
tivity of the targeted drug toward tumor cells can be reliably assessed only
when results with tumor tissue can be compared to those obtained with
normal tissue. Here we describe the use of cultured slices of normal
human brain, obtained by epilepsy surgery, as a control assay for testing
selective cytopathic activity of oncolytic adenoviruses. Methods to evalu-
ate the cytopathic effect in relation to the viability of the brain slices are
studied. Fresh surgical specimens of healthy cerebral cortex acquired dur-
ing surgery for hippocampectomy were cut into slices of 200-m thickness
and kept in 24-well plates in culture medium (Verwer et al., FASEB J. 16
(2002) 54-60). Slices were infected with several different adenoviruses, i.e.,
wild-type Ad5 and replication-defective adenovirus vectors, Ad.CMV.Luc,
and Ad.survivin.Luc (the latter only expressing the luciferase transgene in
cells with active survivin transcription, i.e., tumor cells). Viability of cells
was assessed before and after treatment by using the MTT-derived WST-1
assay (Roche), Live/Dead kit (L/D, Molecular Probes), and cytochrome oxi-
dase activity histochemistry. Our results indicate that wild-type adenovirus
can efciently infect the brain slices. This resulted in a signicant reduc-
tion of viability and energy metabolism, as measured by the L/D kit and
cytochrome oxidase activity, respectively. The WST-1 assay appeared to be
insufciently sensitive. Experiments with Ad.CMV.Luc and Ad.survivin.
Luc show that luciferase expression is signicantly lower when using the
virus with the tumor-specic promoter. The brain slice model is a valuable
tool for assessment of the selectivity of tumor-targeted agents in neuro-
oncology, such as modied adenoviruses. The brain slice model is particu-
larly useful when the targeting strategy of the oncolytic drug is based on
species-specic (human) proteins, causing xenografted animal tumor mod-
els to be of limited use for addressing the question of tumor selectivity. In
the context of oncolytic agents, cytochrome oxidase activity appears to be
the most favorable method to assess viability of brain slices.
32. NOVEL APPROACHES TO METASTATIC ANIMAL
MODELS: A PILOT STUDY FOR BRAIN METASTASIS IN
MICE, 3D TUMOR GROWTH IN CELLULOSE MATRIX, AND
IN VIVO REAL-TIME IMAGING WITH BIOLUMINESCENCE
M. Mut,
1
J. Carpenter,
2
G. Redpath,
2
D. Mullins,
5
C. Slingluff,
4

B. Kundu,
3
E. Farace,
1
M. Lopes,
2
I. Hussaini,
2
and M. Shaffrey
1
;
1
Neurosurgery;
2
Pathology;
3
Radiology Research;
4
Surgery; and
5
Microbiology, University of Virginia, Charlottesville, Virginia, USA
Animal models for brain tumors have provided a signicant insight in
understanding molecular mechanisms of disease progression and develop-
ment of new treatments. Despite the presence of many models available
currently for glial tumors, there is no reproducible model to mimic human
cancers that metastasize to brain in animals. This is a pilot study of a brain
melanoma metastasis model in mice that may re-create the metastatic cas-
cade of cancer. We have chosen melanoma, which is a tumor with high pre-
disposition for metastasizing to brain. Human melanoma cell lines derived
from patients specimens with metastatic melanoma to the brain are har-
vested and cultured (VMM1 and 86). Cells are transfected with luciferase
marker gene and after assessing its expression; cells are xenografted into
two different groups of immunocompromised mice in this pilot study. In
the rst group made up of four animals, tumors embedded into cellulose
matrix (Gelfoam) are injected stereotactically into brain to allow activa-
tion of melanoma cells in brain tissue milieu. In the second group of four
animals, tumors are injected intradermally to re-create the metastatic cas-
cade resulting in brain metastasis. Multiple transfers of cells from xenograft
of the animal, whose tumor metastasizes to brain, into another animal will
ensure continuity of the cycle. Both intradermal and intacranial injection
groups are imaged for luciferase activity as reecting tumor growth and
patterns of metastasis without sacricing the animals. All four animals in
both groups reected tumor growth in their original injection sites with
luciferase activity detected within four weeks. Luciferase bioluminescence
may be imaged with a simple, cooled, charged couple device (CCD) camera
and is an inexpensive, non-invasive screening and sensitive way of assessing
tumor growth even after intracranial injections. Application of the biolu-
minescence techniques provides in vivo, real-time imaging that is used for
the rst time in a brain metastasis model. This is a pilot study that utilizes
novel approaches to metastatic animal models; namely, this is the rst study
to mimic the metastastic cascade to the central nervous system using cancer
cell lines that are predisposed to brain metastasis and to evaluate in vivo,
real-time imaging enabled by luciferase gene transfection into tumor cells.
33. LOCAL TREATMENT WITH AN
IMMUNOSTIMULATORY CPG- OLIGONUCLEOTIDE IN
PATIENTS WITH RECURRENT GLIOBLASTOMA: RESULTS
OF A PHASE I TRIAL
A. Carpentier,
1,2
A. Tibi,
3
M. Richard,
4
L. Capelle,
1
F. Laigle-Donadey,
2

A. Behin,
2
M. Sanson,
2
R. Van Effenterre,
1
and J. Delattre
2
;

1
Neurosurgery,
2
Neurology, Hpital de la Salptrire, Paris;
3
Agence
Gnrale des Equipements et Produits de Sant (AGEPS), Paris;
4
Oligovax; Paris; France
Oligonucleotides containing one or several CpG motifs (CpG-ODN)
display a strong immunostimulating activity, driving the immune response
toward the Th1 phenotype. In preclinical studies, they have shown prom-
ising efcacy when injected locally in several cancer models, including
gliomas. Limursen, a new phosphorothioate CpG-ODN, was administered
intratumorally by convection-enhanced delivery in patients (pts) with recur-
rent glioblastomas (GBMs). Increasing doses were injected, starting at 0.5
mg and escalating to 1, 2, 5, 10 or 20 mg in cohorts of 3 to 6 patients. The
primary objective was to determine the safety prole of intratumoral Lim-
ursen in patients with recurrent GBM. Twenty-four pts were enrolled in the
study. All patients were previously treated with radiotherapy and, in most
cases, with one or several lines of chemotherapy (1 chemotherapy in 10 pts,
2 in 11 pts). At the time of inclusion, the median age was 56 years (range,
2472 years), and median KPS was 80% (range, 60%100%). Two adverse
events were considered related to the procedure, a local hemorrhage along
the catheter track and a pulmonary embolism secondary to the interrup-
tion of a long-term anticoagulant therapy. Adverse effects possibly/probably
related to the studied drug were moderate. Spontaneously regressive grade 3
lymphopenia was reported in 6 patients. Grade 3 nonhematological toxicity
consisted of reversible ALT elevation (2 pts at the highest dose). Six patients
experienced fever above 38C, mainly at higher doses. The fever peaked on
day 3 and disappeared within a few days. Transient worsening of neurologi-
cal conditions was observed at the highest dose in 3 patients. Preliminary
evidence of antitumor activity was suggested with a local response at the
site of injections in 2 patients. Three other patients had a stable disease
for more than 3 months. Updated data for survival will be presented at
the meeting. In conclusion, Limursen was well tolerated in patients with
recurrent GBM, with sides effects mainly limited to transient worsening of
neurological conditions and fever in a few patients. Limursen is now applied
in a multicentric phase 2 study in recurrent glioblastomas.
34. EARLY PHYSIOLOGICAL AND METABOLIC EFFECTS
OF INTRATUMORAL BCNU ON UNTREATED HUMAN
GLIOMAS
M. Jenkinson,
1
E. Smith,
2
K. Kopitzki,
1
S. Vinjamuri,
3
C. Walker,
4
D.
Pietronigro,
5
and P. Warnke
1
;
1
University of Liverpool, Neuroscience,
Liverpool, UK;
2
The Walton Centre for Neurology and Neurosurgery,
Neuro-radiology, Liverpool, UK;
3
Royal Liverpool University Hospital,
Nuclear Medicine, Liverpool, UK;
4
Clatterbridge Cancer Research Trust,
Bebington, UK;
5
Direct Therapeutics Incorporated, New York, USA
DTI-015 (BCNU dissolved in ethanol) utilizes solvent-facilitated per-
fusion for the intratumoral treatment of gliomas. We have investigated
the early biological effects of intratumoral DTI-015 on newly diagnosed,
circumscribed malignant gliomas. Magnetic resonance imaging (MRI)
and spectroscopy (MRS), single photon emission computed tomography
(SPECT), and computed tomography (CT) perfusion studies were used to
assess the effect of DTI-015 on in vivo tumor physiology and metabolism.
Nine patients (2 female, 7 male) with a median age of 58 years (range: 47
70 years) were enrolled into the study. Histological diagnosis was anaplastic
astrocytoma (n 3) and glioblastoma multiforme (n 6). The median Kar-
nofsky performance score was 90. Tumor volume was 13.6 7.6 cm
3
(mean
SD). The volume of DTI-015 injected was 4.3 1.4 ml (mean SD)
with a BCNU dose of 256 82 mg (mean SD). Mean tumoral cerebral
blood ow signicantly reduced within 72 h of DTI-015 injection (paired
t-test; mean reduction 17.3, P 0.001, 95% CI, 10.024.6). Relative cere-
bral blood volume also reduced signicantly (paired t-test; mean reduction
12.5, P 0.017, 95% CI ,2.922.2). There was a signicant reduction in
FDG utilization (paired t-test; mean reduction 0.28, P 0.001, 95% CI,
0.160.41) and thallium uptake (paired t-test; mean reduction 3.26, P
0.001, 95% CI, 1.784.74), with an increase in the Lip1/Cr ratio (Wilcoxon
signed ranks; P 0.034) after DTI-015 injection. The data forms a biologi-
cal basis for understanding the effects of high-dose BCNU on malignant
gliomas. Early effects can be seen on the tumor vasculature and metabo-
lism, resulting in a pattern of ischemic tumor necrosis.
35. IMATINIB MESYLATE (GLEEVEC) PLUS
HYDROXYUREA: AN EFFECTIVE REGIMEN IN THE
TREATMENT OF RECURRENT MALIGNANT GLIOMA:
PHASE 2 STUDY RESULTS
D.A. Reardon,
1
J. Quinn,
1
J. Rich,
1
J. Vredenburgh,
1
A. Desjardins,
1

S. Sathornsumetee,
1
A. Salvado,
2
Z. Nikolova,
2
D. Bigner,
1
and H.
Friedman
1
; Department of Surgery,
1
Duke University Medical Center,
Durham, North Carolina;
2
Novartis Pharmaceutical Corporation, East
Hanover, New Jersey; USA
In this phase 2 study, we evaluated the activity of imatinib mesylate
(Gleevec), an inhibitor of the PDGF receptor tyrosine kinase with anti-
angiogenic activity and the ability to decrease tumor interstitial pressure,
combined with hydroxyurea in the treatment of patients with recurrent
malignant glioma. Eligibility criteria include the following: recurrent malig-
nant glioma; age 18 years; KPS 60% or greater; less than grade 2 intra-
tumoral hemorrhage; adequate hepatic, renal, and bone marrow function.
Hydroxyurea is administered at 500 mg BID while Gleevec is administered
at 500 mg BID for patients on enzyme-inducing anticonvulsants (EIAC;
phenytoin, carbamazepine, and phenobarbital) and at 400 mg QD for those
not on EIAC. Each treatment cycle is 28 days, and patients are evaluated
for response every other cycle. Sixty-four patients have been enrolled to
date, including 32 with recurrent GBM and 32 with recurrent AA/AO. The
median age is 46 (range 21 to 68); 55% are male and 45% are on EIAC.
All patients had prior XRT. The median number of prior chemotherapy
agents was 3 (range, 15), and the median number of prior progressions
was 2 (range, 17). Toxicity has been limited to grade 3 or 4 hematologic
events in 20% and 5%, respectively, grade 3 edema in 8%, and grade 3 LFT
abnormalities in 3%. Among GBM patients, radiographic responses have
been observed in 9%, while 35% have achieved stable disease. Median pro-
gression-free survival (PFS) for patients with recurrent AA/AO and GBM
are 10.9 and 14.4 weeks, respectively. At 6 months, 26.3% of GBM patients
remain progression free. The rate of radiographic response, median PFS,
and 6-mth PFS rate observed in this study among heavily pretreated patients
with recurrent GBM compare favorably to results achieved with temozolo-
mide in rst relapse, indicating that a randomized trial of imatinib mesylate
plus hydroxyurea versus temozolomide is warranted.
36. IMPROVED SURVIVAL OF HIGH- GRADE GLIOMA
PATIENTS AFTER GENE THERAPY WITH AN ADENOVIRAL
VECTOR CONTAINING THE HERPES SIMPLEX VIRUS
THYMIDINE KINASE GENE: A PHASE 2 STUDY
A. Sandmir,
1
M. Vapalahti,
1
A. Immonen,
1
K. Tynnela,
2
H. Hurskainen,
1

R. Vanninen,
3
G. Langford,
4
N. Murray,
4
S. Yla-Herttuala,
5, 6, 7

and M. Vapalahti
7
; Departments of
1
Neurosurgery,
2
Oncology, and
3
Radiology,
4
Department of Molecular Medicine A.I. Virtanen Institute,
5
Department of Medicine, University of Kuopio, Kuopio, Finland;
6
Ark
Therapeutics, Research and Development, London, UK;
7
Gene Therapy
Unit, Kuopio University Hospital, Kuopio, Finland
A phase 2 study was conducted to evaluate the efficacy and safety
of using Herpes Simplex virus thymidine kinase in an adenoviral vector
AdvHSV-tk (Cerepro, Ark Therapeutics Ltd) with intravenous ganciclovir
in malignant glioma patients. This was a single center, randomized, con-
trolled study involving 36 patients with operable primary or recurrent high-
grade glioma. Seventeen patients were randomized to receive AdvHSV-tk
gene therapy (3 10
10
pfu) by local injection into the wound bed at the
time of tumor resection, followed by intravenous ganciclovir, 5 mg/kg twice
daily for 14 days. The control group of 19 patients received standard care
consisting of radical excision. Patients in both groups with primary tumors
received postoperative radiotherapy. AdvHSV-tk therapy increased mean
survival from 39.0 19.7 (SD) in control patients to 70.6 52.9 weeks in
patients in the active group (log-rank regression P 0.0095). Median sur-
vival increased from 37.7 to 62.4 weeks. The percentage increase in mean
survival was 81% and median survival was 65%. The therapy was well
tolerated as assessed by adverse events, clinical chemistry, hematology, and
immunology. There was no evidence of any deterioration in quality of life
or increased use of concomitant medications. AdvHSV-tk gene therapy with
ganciclovir is a new, well-tolerated, potentially effective therapy for oper-
able high-grade glioma.
37. ROLES OF AURORA A MITOTIC KINASE IN THE
DEVELOPMENT OF MALIGNANT GLIOMAS
H. Saya,
1
O. Kobayashi,
1
and J. Kuratsu
2
;
1
Tumor Genetics and Biology;
2
Neurosurgery, Kumamoto University, Kumamoto, Japan
Chromosomal instability and aneuploidy are remarkable hallmarks of
human cancers. In most cancers, high rates of chromosome gains/losses
leading to aneuploidy have been observed. The causes of aneuploidy involve
failure in various critical mitotic events, including centrosome separation,
chromosome alignment, chromosome segregation, and completion of cyto-
kinesis. The error-free mitosis that is important to genomic integrity is regu-
lated by phosphorylation reactions driven by several evolutionarily con-
served serine/threonine kinases, known as mitotic kinases. Mitotic kinases
include cyclin-dependent kinase 1 (Cdk1) and Polo-related, NimA-related,
Aurora-related, and Warts-related kinases. In mammals, three members of
this kinase family, Aurora-A, -B and C, were identied. Recently, obser-
vations have revealed that Aurora-A kinase activity is required for various
events during mitosis, such as G
2
-M transition, centrosome separation,
chromosome alignment and cytokinesis (Hirota et al., Cell 114, 585, 2003;
Kunitoku et al., Dev. Cell 5, 853, 2003; Marumoto et al., J. Biol. Chem.
278, 51786, 2003). Given that not only elevated expression of Aurora-A but
also depletion of Aurora-A leads to mitotic failure and multinucleation, it is
speculated that the proper timing and amplitude of Aurora-A expression is
important for accurate chromosome segregation and delity of chromosome
transmission (Marumoto et al., Nat. Rev. Cancer, in press, 2005). Further-
more, we generated a transgenic mouse model to investigate the involvement
of Aurora-A overexpression in the tumorigenesis and found that elevated
Aurora-A expression induces malignant transformation in the mouse in the
presence of p53 mutation/loss (Zhang et al., Oncogene 23, 8720, 2004).
These ndings indicate that mitotic aberrations induced by Aurora-A over-
expression with p53-dependent checkpoint abnormality are critical factors
for the cancer formation. We have analyzed Aurora-A expression in malig-
nant gliomas and found that it is frequently overexpressed in anaplastic
astrocytomas and glioblastomas. Especially, the Aurora-A overexpression
is well correlated with giant cell formation in those tumors, which is con-
sistent with data obtained in our transgenic mouse model. Our ndings
strongly suggest that aberrant expression of Aurora-A is a high risk factor
for malignant progression of astrocytic tumors.
38. REGULATION OF PI3K SIGNALING AND
TRANSFORMATION BY PTEN C-TERMINAL-
INTERACTING PROTEINS
F. Furnari,
1
K. Okumura,
1
M. Mendoza,
2
M. Zhao,
3
R. Bachoo,
4

R. DePinho,
4
and W. Cavenee
1
;
1
Ludwig Institute-UCSD, La Jolla,
California, USA;
2
UCSD, Biomedical Sciences Graduate Program, La
Jolla, California, USA;
3
Shanghai Institutes for Biological Sciences,
Institute of Biochemistry and Cell Biology, Shanghai, China;
4
Harvard
Medical School, Boston, Massachusetts, USA
The PTEN tumor suppressor gene is frequently mutated in diverse
tumor types, including those of endometrium, breast, prostate, lung, and
high-grade glioma. The protein possesses an amino-terminal catalytic
domain with lipid phosphatase activity which regulates the AKT pathway
through modulation of the second messenger product of PI3K, PI(3,4,5)P3.
While this catalytic region of the PTEN protein, which controls G
1
cell cycle
progression and hence suppresses tumor formation, has been fundamentally
dened, the function(s) of the carboxy-terminal region of the protein remain
largely unknown. To address the issue we searched for PTEN-interacting
proteins by yeast two-hybrid screening using the PTEN carboxy-terminal
domain, which has been reported to contribute to membrane localization
and protein stability as governed by casein kinase II-directed phosphoryla-
tion. Here we report the identication and characterization of two PTEN
interacting proteins, the histone acetyltransferase, PCAF (p300/CBP asso-
ciated factor), and the oncogenic, v-Jun transcriptional target, MSP58 (58-
kDa microspherule protein). The association between PCAF and PTEN
caused increased and direct acetylation of lysine residues (K125 and K128)
within the catalytic cleft of PTEN, a structure required for PI(3,4,5)P3
selectivity. This PCAF-mediated acetylation of PTEN was dependent on the
presence of growth factors. Reduction of endogenous PCAF activity using
shRNA resulted in a loss of PTEN acetylation in response to growth factors
and restored the ability of PTEN to downregulate PI3K signaling. The bio-
logical signicance of these ndings was evidenced by the capacity of PCAF
to acetylate PTEN and to ablate PTEN-mediated suppression of PI3K/AKT
signaling and G
1
cell cycle arrest. The retention of PI3K/AKT signaling and
cell cycle regulatory activities of acetylation-resistant PTEN K125R and
K128R mutants in the presence of enforced PCAF expression supports a
causal relationship. The association of PTEN with MSP58 was mapped to
the MSP58 FHA domain and required PTEN threonine 366, a site reported
to be phosphorylated in vivo. Additionally, we showed that while MSP58
transformed pten
(-/-)
MEF cells, concurrent introduction of wild-type PTEN
caused a dramatic reduction in the number of MSP58-induced colonies.
This inhibition of cellular transformation required interaction with PTEN
since a point mutant of its interaction domain (Thr366Ala) was without
effect. Importantly, transformation inhibition did not, however, require
PTEN to be catalytically active, as its G129R mutant could inhibit MSP58-
driven transformation. Thus, the C-terminal region of PTEN provides novel
biological functions to this tumor suppressor gene in its ability to regulate
its lipid phosphatase activity through interaction with PCAF and in its abil-
ity to regulate cellular transformation through interaction with MSP58.
39. RAS/RAL-PATHWAY ACTIVATION SUPPRESSES CDC42
ACTIVATION/FLIPS EXPRESSION AND SENSITIZES
GLIOMA CELLS TO TRAIL-INDUCED APOPTOSIS
R. Pieper, A. Panner, and M. Berger; UCSF, Neurosurgery, San
Francisco, California, USA
The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)
protein is an attractive therapeutic molecule because it induces apopto-
sis in glioma cells, but not in normal astrocytes. The present study was
initiated to better understand the basis for this tumor selectivity. Normal
human astrocytes; astrocytes immortalized (but not transformed) by ret-
roviral expression of E6, E7, and hTERT; and astrocytes transformed by
expression of E6, E7, hTERT, and mutant V12 H-Ras were assessed by
Western blot for expression of the caspase-8 inhibitor FLIPs, after which
the cells were exposed to TRAIL (01000 ng/ml, 1 h) and the extent of
TRAIL-induced apoptosis was determined by ow cytometry. Immortal-
ized astrocytes were also retrovirally infected with constructs encoding
mutant forms of Ras that selectively activated the Ras- PI3K (C40 Ras),
Ras-Raf (S35 Ras), or Ras-Ral (G37 Ras) pathways, after which the effects
of PI3K, Raf, or Ral activation on cellular transformation; the levels of the
Ras/Ral target cdc42; FLIPs levels; and TRAIL sensitivity were monitored.
While both normal and immortalized human astrocytes were resistant to
TRAIL-induced apoptosis (up to 1000 ng/ml TRAIL) and expressed high
levels of FLIPs, V12 H-Ras-transformed astrocytes exhibited low levels of
FLIPs and underwent apoptosis following exposure to as little as 200 ng/
ml TRAIL. Only expression of a retroviral construct encoding the Ras/
Ral-selective G37 mutant (or introduction of activated Ral itself) trans-
formed astrocytes, suppressed FLIPs levels, and sensitized the astrocytes
to TRAIL-induced apoptosis in a manner comparable to mutant V12 H-
Ras. In the cells expressing either V12 H-Ras or G37 Ras, suppression of
FLIPs levels and enhanced TRAIL sensitivity was associated with suppres-
sion of phosphorylation/activation of the Ras/Ral target cdc42. Conversely,
retroviral introduction of either cdc42 or FLIPs reversed V12 H-Ras/G37
Ras-mediated suppression of FLIPs levels and conferred TRAIL resistance.
These results show that activation of the Ras/Ral pathway not only leads
to cellular transformation, but also to inhibition of cdc42 activation/FLIPs
expression and sensitization of glioma cells to TRAIL-induced apoptosis.
The extent of Ras pathway activation (which has been shown to be propor-
tional to glioma grade) may therefore be helpful in predicting the sensitivity
of gliomas to TRAIL-induced apoptosis.
40. HISTONE DEACETYLASE INHIBITORS, N-BUTYRIC
ACID AND TRICHOSTATIN A, INDUCE CASPASE-
8-DEPENDENT BUT NOT CASPASE-9-DEPENDENT
APOPTOSIS IN HUMAN MALIGNANT GLIOMA CELLS
T. Komata, T. Kanzawa, T. Nashimoto, H. Aoki, S. Endo, T. Kon,
H. Takahashi, S. Kondo, and T. Ryuichi; Brain Research Institute,
Niigata University, Neurosurgery, Niigata, Japan
Histone deacetylase (HDAC) inhibitors have both apoptotic and dif-
ferentiating effects on various tumor cells. However, the mechanisms
underlying the effect of HDAC inhibitors remain unclear. In this study, we
investigated the function of antiproliferative effect of HDAC inhibitors,
N-butyric acid and trichostatin A, on human malignant glioma cell lines,
U251-MG and D54. MTT assay showed dose-dependent inhibition of cel-
lular proliferation in both cell lines. Cell cycle analysis revealed increased
sub-G
1
population in both lines, and G
1
arrest only in U251-MG cells.
Induction of apoptosis was also supported by the occurrence of DNA
fragmentation in tumor cells treated with HDAC inhibitors. Furthermore,
caspase inhibition assay indicated that HDAC inhibitors-induced apopto-
sis was caspase dependent. Interestingly, neither mitochondrial membrane
potential nor the expression of caspase-9 was changed by treatment with
HDAC inhibitors, suggesting the possibility that HDAC inhibitors-induced
apoptosis was not mediated by mitochondrial cell death pathway. On the
other hand, immunoblotting assay conrmed increased expression of cas-
pase-8 in both lines, and elevation of p21 but not of p27 protein in U251-
MG cells following HDAC inhibitor treatment. Taken together, the HDAC
inhibitors, N-butyric acid and trichostatin A, induce caspase-8-dependent
but not caspase-9-dependent apoptosis with or without p21-mediated G1
arrest in human malignant glioma cells.
41. LOW-MOLECULAR-WEIGHT CALDESMON (L- CAD)
AS A NEW SERUM MARKER FOR GLIOMA
P. Zheng,
1
W.C. Hop,
2
P.A.E. Sillevis Smitt,
1
M.J. van den Bent,
1

C.J.J. Avezaat,
1
T.M. Luider,
1
and J.M. Kros
1
;
1
Neuro-oncology
and
2
Epidemiology & Biostatistics,
2
Erasmus M.C., Rotterdam, The
Netherlands
Recent advances in the treatment of gliomas urge for a disease-
specic serum marker more than ever. So far, no candidate glioma marker
has reached the stage of clinical application. In a study using mass spec-
trometry we identied the specic presence of low-molecular-weight calde-
smon (l-CaD) in cerebrospinal uid (CSF) samples of glioma patients. We
subsequently showed that l-CaD is specically present and overexpressed in
glioma vasculature but not in the blood vessels of normal brain. Additional
RT-PCR experiments to microdissected components of glioma samples
provided evidence that three out of four splice variants of the CALD1 gene
are specically expressed in the glioma vasculature. Therefore, l-CaD may
be regarded as a glioma-specic marker. Here we tested the feasibility of
using l-CaD as a serum marker for glioma. A total of 230 serum samples,
including sera of 57 patients with glioma, 107 patients with intracranial
tumors other than glioma including metastases, 36 patients with various
neurological diseases but no tumors, and 30 healthy subjects were tested
for the l-CaD protein level by ELISA. The specicity of the assay was moni-
tored by immunoprecipitation and immunoblotting (IP/IB). The serum level
of appeared to be signicantly higher in the group of glioma patients than
in any of the control groups (pl-CaD levels between patients with low-
or high-grade gliomas. The serum l-CaD level as determined by ELISA is
a good discriminator between glioma patients versus patients with other
intracranial tumors, other neurological diseases, and healthy people. It does
not, however, provide information as to histological malignancy grade of
the glioma. Prospective studies are initiated to test the contribution of the
assay in making the diagnosis of glioma, or its feasibility for monitoring
disease during treatment.
42. HIGH POSITIVE PREDICTIVE VALUE OF HEMIZYGOUS
DELETIONS AT THE NOTCH2 LOCUS FOR SURVIVAL OF
BRAIN TUMOR PATIENTS
B. Sivasankaran,
1
B. Jean-Louis,
1
I. Mihai CS,
1
T. Elisabeth,
1
D.
Beatrice,
1
G. Anthony,
2
Z. Christian,
2
M. Adrian,
2
M. Andr R,
3
and
R. Guido
4
;
1
Neuro Oncology and
2
Neurosurgical Clinic, University
Hospital, Basel, Switzerland;
3
University Hospital, Department of
Internal Medicine, Bruderholz, Switzerland;
4
Heinrich-Heine-University,
Department of Neuropathology, Dsseldorf, Germany
Loss of heterozygosity (LOH) on chromosome 1p predicts responsive-
ness to chemotherapy in 70% of malignant oligodendrogliomas (OGs),
pointing to a genetic factor for the response that distinguishes OGs from
resistant glioblastomas (GBMs). We dened eight distinct haplotypes on a
somatic deletion map on chromosome 1 of 26 OGs and 50 GBMs. In search
for a correlation between survival and particular haplotypes, factor analy-
sis, multivariate analysis, and non-parametric Kaplan-Meier curves were
used. Test accuracy was determined by receiver operating characteristic
(ROC) analyses. A consistent centromeric recombination breakpoint, clus-
tered within 1 centiMorgan between markers D1S2696 and D1S2344, was
prevalent in OGs, but not in GBMs (P 0.0001). Hemizygous deletions
at D1S2696, located within intron 12 of the Notch2 gene, correlated with
better outcome (P 0.0001). Interestingly, primary OGs and a subgroup
of GBMs harbored overlapping single-copy microdeletions, dening a mini-
mally lost region of 45 kb within the coding sequence of the Notch2 gene.
LOH at the marker D1S2696 denes a new molecular classication that is
independent of patient age and gender and equally well predicts survival
time as histological and immunohistochemical classication (P 0.0001).
By ROC analysis, a cut-off of 24 months of survival time was dened result-
ing in a sensitivity and specicity for molecular classication of 80.8%
and of 92.0%, respectively (positive and negative predictive value 84.0%
and 90.2%). Simple, rapid, and highly reproducible molecular classication
using marker D1S2696 at the Notch2 locus adds independent prognostic
information to histological classication and identies a subgroup of OG-
like GBMs with long-term survival.
43. GENE EXPRESSION PROFILING LINKS INVASION-
RELATED GENES TO POOR SURVIVAL IN OLDER
GLIOBLASTOMA PATIENTS
J. Rich,
1
C. Hans,
2
B. Jones,
2
R. McLendon,
3
B. Rasheed,
3
A. Dobra,
2

H. Dressman,
4
D. Bigner,
3
J. Nevins,
4
and M. West
2
;
1
Department of
Medicine
2
Institutes of Statistics and Decision Sciences, and Departments
of
3
Pathology and
4
Molecular Genetics & Microbiology, Duke University
Medical Center, Durham, North Carolina, USA
Despite the strikingly grave prognosis for older patients with glioblasto-
mas, signicant variability in patient outcome is experienced. To explore the
potential for developing improved prognostic capabilities based on the eluci-
dation of potential biological relationships, we performed analyses of genes
commonly mutated, amplied, or deleted in glioblastomas and Affymetrix
DNA microarray gene expression data from tumors of 43 glioblastoma
patients of age greater than 50 for whom survival is known. No prognostic
signicance was associated with genetic changes in EGFR, TP53, p16
INK4A
,
or PTEN. Statistical analysis of the gene expression data in connection
with survival involved exploration of regression models on small subsets
of genes, based on computational search over multiple regression models
with cross-validation to assess predictive validity. The analysis generated
a set of regression models that, when weighted and combined according to
posterior probabilities implied by the statistical analysis, identify patterns
in expression of a small subset of genes that are associated with survival and
have value in assessing survival risks. The dominant genes across multiple
such regression models involve three key genes, secreted protein acidic and
rich in cysteine (SPARC, osteonectin), Doublecortex and Semaphorin3B,
which play roles in cellular migration processes. Additional analysis, based
on statistical graphical association models constructed by using similar
computational analysis methods, reveals others genes that support the view
that multiple mediators of tumor invasion may be important prognostic fac-
tors in glioblastomas in older patients. No previous studies of which we are
aware have elucidated conclusive links between expression of specic gene
and survival of older glioblastoma patients. Our regression analyses using
gene expression as explanatory of survival outcomes revealed that genes
whose primary cellular effects may be the regulation of cellular migration
appear as candidate markers of poor survival. Together these results suggest
that tumor migration may represent an important effector of glioblastoma
malignancy and may warrant accelerated development of specic therapies.
Future studies will prospectively determine the link between the expression
of SPARC, doublecortex, and SEMA3B in gliomas of all grades and patient
outcome. This work was supported by a grant from the W.M. Keck Foun-
dation. J.N.R. is a Damon Runyon-Lilly Clinical Investigator and a Sidney
Kimmel Cancer Foundation Scholar. This work was also supported by NIH
grants NS047409 (J.N.R.).
44. CRITICAL ANALYSIS OF THE WHO
HISTOPATHOLOGICAL GRADING FOR MENINGIOMAS.
IMPACT ON POSTOPERATIVE RADIOTHERAPY AND
FOLLOW UP IN GERMANY
M. Simon, J. Bostrm, P. Koch, and J. Schramm; University Hospital
Bonn, Department of Neurosurgery, Bonn, Germany
We critically analyzed the clinical impact of the WHO histological
grading for meningiomas including its role for postoperative radiotherapy/
radiosurgery indications and MR follow-up protocols. The current (2000)
and the 1993 WHO classications were used to review the histological
grade of 57 meningiomas operated at our institution. All German Neuro-
surgical Departments performing intracranial microsurgery were asked to
detail their guidelines for radiation therapy and follow-up for meningiomas
of different WHO grades. Comparing both WHO classications, the cur-
rent criteria downgrade 7/15 (47%) atypical (WHO grade II, MII) menin-
giomas to grade I (MI) and 4/6 (67%) anaplastic (WHO grade III, MIII)
tumors to grade II. The use of specic criteria to diagnose atypia (MIB1
index 5%) and malignancy (brain invasion) only during the rst review
accounted for 3 grade II to I and all 4 grade III to II reclassications, respec-
tively. Indications for radiation therapy and MR follow-up protocols varied
substantially with the histological grade and between institutions. After an
incomplete resection, radiotherapy recommendations differed between MI
and MII in 30/58 (52%), and between MII and MIII in 34/56 (61%) units.
Our data document a considerable impact of the histological grading for
meningiomas in clinical practice. However, the use of changing grading
paradigms in recent years renders clinical decision making based on local
and published experience difcult. The categories atypical and anaplastic
meningioma, WHO grade II and III, respectively, have probably described
quite different tumors in recent years. The clinical relevance of meningioma
grading will only be properly recognized if diagnostic neuropathological
labels are used consistently.
45. NOVEL STRATEGIES OF IMMUNOTHERAPY FOR
MALIGNANT GLIOMA
M. Weller; University of Tubingen, Neurologische Klinik, Tubingen,
Germany
The limited efcacy of surgery, radiotherapy, and chemotherapy in the
treatment of malignant glioma calls for innovative treatment approaches
targeting specic biological features of these tumors. Malignant glioma
cells have long been known for the release of multiple established or puta-
tive immunosuppressive molecules, including transforming growth factor
(TGF)-beta, prostaglandins, interleukin 10, CD95 ligand, or HLA-E/G.
The very low frequency of systemic metastases in tumors which otherwise
exhibit all features of malignancy has been attributed to an efcient glioma
immune surveillance outside, but not inside, the central nervous system.
Accordingly, promising strategies of immunotherapy based on these obser-
vations include (i) targeting the synthesis, release, or activity of glioma-
derived immunosuppressive molecules and (ii) taking the presumably effec-
tive immune surveillance from the outside into the central nervous system.
Paradigmatically, TGF-beta, the prime suspect for glioma-associated
immunosuppression, may be antagonized by RNA interference, inhibition
of proprotein processing or TGF-beta receptor antagonists, conferring
resistance to TGF-beta-induced immune paralysis. In fact, while the bio-
logical neutralization of glioma-associated immunosuppressive molecules
alone may not induce the immune rejection of these tumors, it may still
be a precondition for active strategies of immunotherapy to be successful.
Until specic tumor antigens for malignant glioma may be identied, the
most promising strategies of immunotherapy include those based on the
vaccination with autologous, genetically modied tumor cells in the context
of a stimulatory immune environment, possibly employing dendritic cell
immunity. Future experimental trials in glioma-bearing rodents and phase
1/2 clinical trials will have to demonstrate whether the initiation of an ef-
cient efferent immune response against malignant glioma cells may be more
easily triggered by peripheral vaccination or by efforts to create a strong
immune stimulatory environment within a postsurgical tumor cavity.
46. PSEUDOTYPED, ONCOLYTIC ADENOVIRUSES USED TO
TARGET GLIOMA STEM CELLS
J. Rebetz,
1,2,3
A. Edqvist,
1,2,3
L. Geironson,
1,2,3
H. Liu,
1,2,3
L.G. Salford,
1

X. Fan,
1,2,4
and B. Widegren
1,2,3
;
1
The Rausing Laboratory, Division
of Neurosurgery,
2
Section of Immunology,
3
Division of Genetics,
Department of Cell and Molecular Biology, and
4
Strategic Research
Center for Stem Cell Biology and Cell Therapy, Lund University, Lund,
Sweden
We have developed a number of oncolytic viruses based on a controlled
gene expression by the human telomerase revere transcriptase promoter.
We describe ve different oncolytic viruses: (1) an adenovirus using the
CMV promoter to express the herpes simplex virus thymidine kinase gene
(HSV-TK) in combination with Gancyclivir, (2) an adenovirus using the
hTERT promoter to express the HSV-TK gene in combination with Gan-
cyclivir, (3) an adenovirus using the hTERT promoter to express the cyti-
dine deaminase gene fused to a uracil kinase gene in combination with 5-F-
deoxycytidine, (4) an adenovirus using the hTERT promoter to express the
adenoviral E1 proteins for replication in and lysis of hTERT positive cells,
and (5) an adenovirus using the hTERT promoter to express a modied
drosophila cytidine kinase, developed by ZGENE (Denmark) in combina-
tion with Gemcitibine. All of the hTERT promoter-containing viruses have
an upstream chicken insulator element to prevent activation by cis-acting
enhancer/promoters. This insulator element increases the specicity and
integrity of the hTERT promoter. The proof of concept about this promoter
construct is available (Edqvist et al. ibid). The adenovirus we use is a pseu-
dotyped adenovirus that uses the CD46 molecule as the receptor instead of
the coxsackie adenovirus receptor (CAR). The knob and the shaft of the
ber protein come from adenovirus type35. We will test these viruses on at
least 20 different primary glioma cell cultures and xenografted subcutane-
ously growing gliomas. The frequency of hTERT positive cells before and
after the treatment will be evaluated. Preliminary data will be given and
discussed.
47. ANTISENSE-MEDIATED SUPPRESSION OF HEPARANASE
GENE INHIBITS BRAIN METASTASIS OF MELANOMA
CELLS
M. Roy,
1
J. Reiland,
1
B.P. Murry,
1
V. Chouljenko,
2
K.G. Kousoulas,
2
and
D. Marchetti
1
;
1
Department of Comparative Biomedical Sciences and
2
Division of Biotechnology & Molecular Medicine, School of Veterinary
Medicine, Louisiana State University at Baton Rouge, Baton Rouge,
Louisiana, USA
Brain metastasis occurs in most of all human cancers and is a frequent
manifestation of malignant melanoma progression. Successful invasion into
brain by tumor cells must include attachment to microvessel brain endo-
thelial cells, and, of relevance, degradation of surrounding extracellular
matrix (ECM). Heparan sulfate proteoglycans (HSPG) are essential and
ubiquitous macromolecules associated with the cell surface and ECM of a
wide range of cells and tissues. Heparanase (HPSE-1) is an ECM degrada-
tive enzyme and a critical molecular determinant in metastatic events. The
enzyme acts as an endo-beta-d-glucuronidase which degrades the heparan
sulfate (HS) chains of HSPG at specic intrachain sites, resulting in bioac-
tive HS fragments of discrete molecular weight size. To investigate effects
of changes in heparanase gene expression in brain-metastatic melanoma
(BMM) cells, we constructed adenoviral vectors containing the full-length
human HPSE-1 cDNA in both sense (Ad-S/hep) and antisense orientation
(Ad-AS/hep). We found increased HPSE-1 expression and activity in BMM
following Ad-S/hep infection by Western blot analyses and specic HPSE-
1 activity assays. Conversely, HPSE-1 content was signicantly inhibited
following infection with Ad-AS/Hep. Importantly, HPSE-1 modulation by
these adenoviral constructs correlated with brain invasive cellular proper-
ties in vitro. Moreover, extensive brain metastasis formation was observed
in athymic nu/nu mice injected with Ad-S/hep-infected BMM cells, while
none of the mice injected with Ad-AS/hep showed any evidence of macro-
scopic malignancy. Our results suggest that HPSE-1 not only contributes
to the brain-metastatic phenotype of melanoma cells, but also that the Ad-
AS/hep-mediated inhibition of its enzymatic activity can be efcacious in
the prevention and treatment of melanoma brain metastasis.
48. ADENOVIRAL VECTOR MEDIATED DETECTION OF
TELOMERASE ACTIVITY AT SINGLE LIVING CELLS
A. Edqvist,
1
J. Rebetz,
1
M. Jrs,
2
L. Salford,
3
X. Fan,
1,2
and B.
Widegren
1,3
;
1
2
Section of Molecular Medicine
and Gene Therapy, and
3
The Department of Neurosurgery, The Rausing
Laboratory, Lund University, Lund, Sweden
Telomerase activity is normally observed in highly proliferative tissues,
stem cells, and most malignant cells and therefore offers an attractive target
for therapeutic intervention and for diagnostic and prognostic purposes.
The telomerase activity is predominantly controlled by the regulated expres-
sion of the catalytic subunit telomerase reverse transcriptase (hTERT) at the
transcriptional level. Here, we developed adenoviral vectors for detecting
telomerase activity in single living cells. In these vectors, the expression
of destabilized enhanced green uorescence protein with a half-life of 2
h (d2EGFP) is under the control of the hTERT promoter. Insulator DNA
sequences were introduced to shield the hTERT promoter from cis-activat-
ing elements in the adenoviral vector backbone. Moreover, the vectors were
retargeted to ubiquitously expressed CD46 as a cellular receptor. Following
infection of telomerase positive (HeLa and A549 cells) or negative cells
(broblast and WI-38 cells) with such vectors, the d2EGFP expression was
detected in a telomerase activitydependent manner, which correlated with
the hTERT expression as assessed with real-time PCR. Furthermore, about
50% of the promyelocytic leukemic HL-60 cells were expressing d2EGFP
following infection with telomerase reporting adenoviral vector, and the
d2EGFP expression was signicantly diminished in retinoic acidinduced
differentiating HL-60 cells compared with nontreated control cells. Cell
cycle analysis showed that the sorted d2EGFP HL-60 cells were mostly
in the S/G
2
/M phase of cell cycle, whereas the d2EGFP- HL-60 cells mainly
at the G
1
and early S phase. In addition, our telomerase reporting vectors
allowed high levels of d2EGFP expression in early passage xenograft glioma
cells and the percentage of d2EGFP expression glioma cells diminished in
late passages. Thus, the dynamically regulated telomerase activity during
cell proliferation and differentiation in single living cells can be identied
following infection with our adenoviral reporting vectors. Our studies pro-
vide a powerful tool for identifying living cells with telomerase activity.
49. ROLE FOR C-JUN N-TERMINAL KINASE (JNK) IN
RAS-MEDIATED NON-APOPTOTIC PROGRAMMED
CELL DEATH
W. Mouri, T. Kayama, and C. Kitanaka; Yamagata University,
Neurosurgery, Yamagata, Japan
Accumulating evidence now indicates that nonapoptotic, caspase-
independent mechanisms play a critical role in oncogene- as well as anti-
cancer-treatment-induced cellular suicide. We have found that the ras
oncogene induces nonapoptotic programmed cell death in glioma and neu-
roblastoma cells, but the molecular mechanism involved therein remains
undened. Cell death was induced by transient cotransfection of active Ras
(H-RasV12)- and green uorescence protein (GFP)-expressing vectors and
was assessed on the basis of morphology of GFP-positive cells. Ras and
JNK protein expression levels were determined by immunoblotting with
specic antibodies for each. Activation of JNK was monitored by immuno-
blot analysis using a JNK antibody that specically recognizes JNK phos-
phorylated at Thr183/Tyr185. Blockade of JNK activity was achieved by
the use of SP600125, a highly specic chemical inhibitor of JNK. Increase
of phosphorylated (active) JNK was detected in the course of Ras-induced
nonapoptotic cell death. Dose-dependence analysis showed that increased
expression of Ras (via transfection of increasing amounts of Ras expression
vector) is associated with increased JNK activity, yet an inactive Ras mutant
(H-RasN17) failed to activate JNK as well as cell death itself. Time-course
analysis demonstrated that JNK activation followed Ras expression and
preceded cell death. SP600125 inhibited Ras-induced nonapoptotic cell
death in a range of concentrations where it efciently inhibited JNK activ-
ity. The results indicate that (i) Ras activates JNK during cell death induc-
tion, depending on its ability to transduce intracellular signals and that (ii)
JNK likely has a causal role in triggering nonapoptotic cell death. Thus the
data together suggest that JNK is involved in the death signal transduction
of Ras-mediated nonapoptotic programmed cell death.
50. THE MOST CONSTITUTIVELY ACTIVE JNK
ISOFORM, JNK2A2, IS PREFERENTIALLY EXPRESSED
IN GLIOBLASTOMAS: IDENTIFICATION OF SPECIFIC
SEQUENCES LEADING TO ITS ACTIVATION
J. Wong and J. Cui; Thomas Jefferson University, Kimmel Cancer
Institute, Philadelphia, Pennsylvania, USA
C-jun N-terminal kinases (JNKs) are critical to regulating cell growth,
proliferation, and apoptosis. Activation of the JNK pathway has been
implicated in the formation of several human tumors, particularly gliomas.
There are 10 different JNK isoforms. We previously showed that a 55KD
JNK isoform is constitutively activated in 86% of human gliomas, which
makes it the most frequent signaling alteration found in these tumors. We
found that this isoform was specically a JNK2 isoform and likely to be
either JNK2a2 or JNK22. Notably, we showed that JNK2a2 possesses the
strongest autophosphorylation activity among all isoforms. We now report
our efforts to identify specic sequences that contribute to JNK2a2 activa-
tion and how this isoform contributes to glial tumorigenesis. We generated a
series of chimeric cDNAs that join portions of JNK1a2, which lacks detect-
able autophosphorylation activity, with portions of JNK2a2, which has the
strongest activity. Through in vivo and in vitro kinase assays, we dened a
domain within JNK2a2 from amino acid 218 to 226 that is required for its
autophosphorylation. Mutation of JNK2a2 to its counterpart of JNK1a2 in
this region abrogated the autophosphorylation activity and c-jun substrate
kinase activity in vivo and in vitro. The switching of JNK1a2 to JNK2a2
at this region enabled JNK1a2 to gain autophosphorylation activity. Next,
we examined the expression of JNK1a2, JNK2a2, JNK12, and JNK22
in normal brain specimens and glioblastomas by RT-PCR. All four isoforms
were expressed in normal brains (3/3). JNK1a2, JNK12, and JNK22
were found in only 18% (2/11) of these tumors. In contrast, JNK2a2 was
found in 91% of glioblastomas (10/11). We then transfected U87-MG cells
with GFPC1-JNK1a1, GFPC1-JNK2a2, and GFPC1- JNK2a2/APF (a
dominant negative mutant form of JNK2a2) and obtained stable clones
expressing similar levels of protein. We assessed the effects on parameters
related to tumorigenesis including cell proliferation, soft agar colony for-
mation, and tumor formation in athymic mice. JNK2a2 was consistently
the most effective in promoting proliferation and tumor growth where the
relative order was JNK2a2 JNK1a1 JNK2a2/APF. Since JNK activates
transcription factors, we proled gene expression using cDNA microarrays.
There were 16 genes whose expression was upregulated by JNK2a2 but sup-
pressed by JNK2a2/APF, including EIF-4E and TGF-a, which indicates that
these genes may be critical for JNK2a2 effects on transformation. Our data
indicate that glioblastomas specically upregulate the most active JNK iso-
form which promotes tumorigenesis through upregulation of specic genes.
The identication of specic sequences that lead to JNK2a2 activation will
allow us to design specic inhibitors.
51. ESTABLISHMENT OF A CELL LINE DERIVED FROM
HUMAN CENTRAL NERVOUS SYSTEM LYMPHOMA
S. Fujita, J. Ueda, H. Ikemoto, K. Mori, and N. Arita; Hyogo College of
Medicine, Neurosurgery, Nishinomiya, Hyogo, Japan
Primary central nervous system lymphoma (PCNSL) exhibits several
characters different from systemic lymphoma cells. PCNSL are usually con-
ned to the central nervous system, vanish completely when treated with
steroids, anticancer drugs or radiation, but relapse very rapidly. Little has
been reported, however, on the genetic and biological nature of PCNSL,
because of the scare tissue specimens derived from stereotactic biopsy. We
established a cell line from human PCNSL and aimed to inquire its charac-
ter of PCNSL. The cell line was established from the surgical specimen of
PCNSL in the right putamen of a 68-year-old female, using primary explant
technique. We investigated the character of lymphoma cells by immunocy-
tochemistry, electron microscopic observation, and immunoblot analysis
and at the same time, studied the response of cells against dexamethasone
and methotrexate (MTX) in vitro. Apoptosis was analyzed by the Tunel
method. To check the genetic changes, spectral karyotyping was performed.
The population-doubling time of the cultured cell was 20 h. The cultured
cell in RPMI1640 supplemented with 10% fetal bovine serum reacted with
anti CD20, BCL2, and BCL6 antibodies. Electron microscopic observation
revealed nuclear bleb, which was specic for lymphoma cell. Immunoblot
analysis revealed BCL-2 and BCL-6 are expressed in the cultured cell. Rep-
resentative karyotype was interpreted as 50,XX, 3(5), t(4;15)(q31;q15),
del(6)(q21q25),18(10). Inhibition dose (ID50) of MTX was 6 nM, and
ID50 of dexamethasone was 2 nM. Apoptosis was detected after either
MTX or dexamethasone treatment. Our results demonstrated that the
established cell line (designated MCL2) could be the useful in vitro model
of PCNSL.
52. THE MITOCHONDRIAL PATHWAY IS CENTRAL TO
ERUCYLPHOSPHOCHOLINE-MEDIATED APOPTOSIS IN
HUMAN GLIOMA CELL LINES
W. Kugler, F. Khler, F. Buchholz, B. Erdlenbruch, and M. Lakomek;
Universitts-Kinderklinik, Pdiatrie I, Gttingen, Germany
Glioblastomas are highly chemo- and radioresistant tumors of the CNS.
Therefore, new therapeutic approaches are urgently needed. Alkylphospho-
cholines (APCs) are membranophile agents not directly targeting cellular
DNA. Erucylphosphocholine (ErPC) represents the prototype of a promis-
ing class of APC for parenteral administration. It has potent antineoplastic
activity on various malignant tumors of different origin and accumulates
within the brain. Of particular interest, ErPC induces apoptosis in glioma
cells resistant to treatment with standard chemotherapeutics. Recently, we
have shown that ErPC mediates apoptosis independent of p53 signaling and
death receptor/ligand systems, whereas activation of caspases via mitochon-
dria plays a major role. To analyze the contribution of the mitochondrial
pathway in more detail, we investigated the cytotoxic effects of ErPC in
tumor cells with defects in apoptosis. To this end, we silenced the expres-
sion of Apaf-1 and caspases-3 and -9 in human glioma cell lines by specic
small interfering RNAs (siRNAs). The different siRNAs showed a vari-
able degree of knockdown efciency with respect to protein expression and
induction of apoptosis arguing in favor of an essential role of the intrinsic
pathway. As a further proof that the poor response to ErPC is due to the
lack of an essential apoptosis gene, we used HeLa cells stably transfected
with a dominant-negative caspase-9 construct, Apaf-1-negative melanoma
cells, and MCF-7 cells harboring a deletion in the Casp-3 gene. In these
cell lines, cytotoxicity and apoptosis induction was drastically decreased
compared to vector controls, to cells harboring a functional Apaf-1 gene,
and to cells stably transfected with the Casp-3 cDNA, which thus corrobo-
rated our results in glioma cells. In particular, we provide evidence that
caspase-3 is required for the activation of caspases-2, -6, and 8 and also
participates in a feedback amplication loop. Together, our data suggest
that components of the mitochondrial apoptosis pathway are essential for
ErPC to effectively induce apoptosis, whereas elements of the death receptor
pathway are dispensable. This work was supported by B. Braun-Stiftung.
53. TRAIL INDUCES PROLIFERATION OF MALIGNANT
GLIOMA CELLS THROUGH C-FLIP-MEDIATED ERK1/2
PATHWAY
S. Vilimanovich,
1
J. Song,
2
L. Guilbert,
1
and C. Hao
2
;
1
University of
Alberta, Laboratory Medicine & Pathology and Medical Microbiology
& Immunology, Edmonton, Canada;
2
Emory University, Pathology &
Laboratory Medicine, Atlanta, Georgia, USA
Tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL)
induces apoptosis in TRAIL-sensitive human malignant glioma cells. Here
we show that TRAIL stimulated cell growth in TRAIL-resistant glioma
cells. TRAIL-induced cell growth in the resistant cells occurred through
increased cell cycle progression as determined by ow cytometry analysis of
propidium iodidestained cells and Western blot analysis of retinoblastoma
protein (pRb) phosphorylation. Western blot analysis of TRAIL-treated
resistant cells revealed phosphorylation of ERK1/2 proteins, and in vitro
kinase analysis conrmed the activation of the ERK1/2 kinases. ERK1/2
kinases are activated through dual phosphorylation by mitogen-activated
protein kinase (MAPK)/ERK kinase (MEK). Treatment of the resistant
cells with MEK1 inhibitor PD98059 eliminated TRAIL-induced ERK1/2
activation and cell proliferation. These results suggested that TRAIL-
induced cell proliferation occurs through activation of the ERK1/2 path-
way in TRAIL-resistant glioma cells. Inhibition of cellular Fas-associated
death domain-like interleukin-1-converting enzyme-inhibitory protein
(c-FLIP) with small interfering RNA (siRNA) eliminated TRAIL-induced
ERK1/2 activation, and proliferation as determined by cell viability assay,
pro pidium iodide staining, and ow cytometry, and pRb phosphorylation.
The results indicate that TRAIL-induced ERK1/2 activation and prolifera-
tion in TRAIL-resistant glioma cell lines is dependent upon the expression
of the caspase-8 inhibitor c-FLIP. Furthermore, inhibition of c-FLIP expres-
sion sensitized the resistant cells to TRAIL-induced apoptosis as demon-
strated by the cleavage of caspases. In conclusion, TRAIL triggers growth in
TRAIL resistant malignant glioma cells through c-FLIP-mediated ERK1/2
pathway, and thus targeting c-FLIP-ERK1/2 pathway may overcome the
resistance of malignant glioma cells to TRAIL treatment.
54. ROCK INHIBITION INDUCES ASTROCYTOMA
MOTILITY IN A RAC1-DEPENDNET MANNER
B. Salhia, F. Rutten, and J. Rutka; The Hospital for Sick Children, Brain
Tumour Research Center, Toronto, Canada
The intracellular mechanisms governing astrocytoma motility while
poorly understood require modications of the cytoskeleton. The Rho-
GTPases (Rac, Cdc42 and Rho) are pivotal regulators of cytoskeletal
organization and cell motility. We have shown that inhibition of ROCK, a
serine/threonine effector kinase of Rho, with Y27632 or the dominant-neg-
ative mutant inhibits stress bers and focal adhesions induced by LPA. In
contrast to several studies demonstrating that inhibition of ROCK leads to
decreased tumor cell invasiveness and motility, we found using a 2-dimen-
sional radial migration assay that astrocytoma migration was signicantly
increased following treatment with Y27632. LPA also signicantly stimu-
lated the motility of astrocytoma cells. The observation that ROCK inhibi-
tion also led to increased membrane rufing suggested that Rac activation
was a possible mechanism in Y27632-induced motility. Rac-GTPases are
thought to regulate membrane rufing formation and cell migration in large
part by stimulating actin polymerization. We demonstrated for the rst time
both directly and indirectly that Rac activation is an outcome of ROCK
inhibition in astrocytoma cells. First, using a Rac-GTP pull-down assay,
we show that U251 cells treated with Y27632 increased Rac activity com-
pared to untreated controls. In addition, LPA alone or in combination with
Y27632 also increased the levels of Rac-GTP. Next we show that Y27632
induces membrane rufing in a Rac1-dependent manner since depletion
of Rac1 strongly inhibits Y27632-induced membrane rufes. These cells
also reverse the stellate phenotype of Y27632 treatment without regaining
actin stress bers. In addition, Rac1-directed siRNA effectively overcame
Y27632-induced motility by nearly 2-fold. Furthermore, Rac1 depletion
alone inhibited the migration of U251 cells by about 50%. In summary,
our data show that inhibition of ROCK plays a major role in regulating
astrocytoma cell morphology, actin cytoskeleton, and migration through
the activation of Rac1. In addition, astrocytoma migration seems to be
occurring by two separate, Rho-independent, Rac-dependent mechanisms,
ROCK inhibition and LPA stimulation. Our future studies will be directed
toward determining the mechanisms that lead to Rac activation following
ROCK inhibition or LPA stimulation in our cells. Increasing our under-
standing of the signicant cross-talk that appears to be occurring between
Rho-GTPase family members and their effector proteins will be important
to identifying key elements within these pathways that can be exploited to
inhibit the growth and invasiveness of human astrocytoma cells.
55. MEMBERS OF THE ETS FAMILY OF TRANSCRIPTION
FACTORS BIND TO THE SITE INTRODUCED BY A SINGLE
NUCLEOTIDE POLYMORPHISM IN THE MMP-1 PROMOTER
J. McCready,
1
V. Sykes,
2
W. Broaddus,
2
and H. Fillmore
2
;
1
Anatomy
and Neurobiology;
2
Neurosurgery, Virginia Commonwealth University,
Richmond, Virginia, USA
Matrix metalloproteinase-1 has been implicated in the metastasis and
invasion of many types of tumors. A polymorphism exists in the proximal
promoter region of MMP-1 that regulates MMP-1 transcription. This poly-
morphism consists of either the presence (2G allele) or absence (1G allele)
of a guanine nucleotide adjacent to a pre-existing guanine nucleotide. This
additional guanine nucleotide creates a binding site for the ETS family of
transcription factors and leads to increases in MMP-1 transcription. In sev-
eral aggressive and metastatic tumors studied, the incidence of the 2G allele
is signicantly higher. We found a signicant difference in the distribution
of the genotypes between healthy individuals and glioblastoma patients (P
0.031) with an increase in the percentage of the 2G/2G genotype in the
tumor population (P 0.018). The aim of this study was to determine if
the additional ETS binding site regulates the MMP-1 promoter in glioma
cells. Transfection of three glioma cell lines with a 2G MMP-1 promoter
reporter construct results in increased transcription when compared to
transfection with the 1G reporter construct (P 0.02). Identication of
the proteins binding to the 2G promoter is the rst step in understanding
the regulatory effects this polymorphism has on MMP-1 transcription. ETS
transcription factors are divided into subfamilies according to structural
similarities. Results from RT-PCR and Western blot indicate that all mem-
bers of the Ets and Pea3 subfamilies are present in seven glioma cell lines.
To determine which members of these subfamilies bind to the 2G promoter,
we performed DNA-protein pull-down assays. Our data indicates that Ets-1
binds to the 2G promoter, and we are currently evaluating the binding capa-
bility of the other members of these subfamilies. We are also conducting
chromatin immunoprecipitation assays to determine which proteins bind
to the 2G allele in the context of the cellular environment. Both hepatocyte
growth factor and phorbol myristate acetate have been shown to increase
binding of ETS members to the MMP-1 promoter leading to increases in
MMP-1 promoter activity. We are assessing the ability of HGF and PMA
to inuence the binding of ETS proteins to the 2G promoter and subsequent
MMP-1 transcriptional activity. Results from these studies will increase our
knowledge of how MMP-1 is regulated in glioma cells. This information
may lead to advances in therapies that articially lower the levels of MMP-1
and indirectly control glioma invasion.
56. REGULATION OF UNCOUPLING PROTEIN-2 (UCP-2)
EXPRESSION IN HUMAN GLIOMA CELLS BY PEROXISOME
PROLIFERATOR-ACTIVATED RECEPTOR (PPAR) AGONISTS
J. Brown, J. Darling, S. Dunmore, and S. Bassey; University of
Wolverhampton, RIHS, Wolverhampton, UK
Recent studies have suggested that the glitazones, a group of PPAR ago-
nists commonly prescribed as therapy in type 2 diabetes, could have a role
in regulation of cell viability in astrocytomas and glioma cell lines, possibly
due to a modulation of reactive oxygen species (ROS) production. PPAR
agonists are also known to regulate expression of the mitochondrial protein
UCP-2, and UCP-2 has a purported role in ROS regulation amongst others.
This study investigated the expression of UCP-2 in U251MG glioma cells
and its regulation by PPAR agonists. U251MG glioma cells were cultured
according to standard methods and treated with PPAR alpha, delta, and
gamma agonists (10 M WY14643, 10 nM PGI2, 10 M rosiglitazone and
10 nM PGJ2, respectively) for 24 h. Total RNA was subsequently extracted
and semiquantitative RT-PCR used to evaluate UCP-2 mRNA expression.
Results showed that UCP-2 was expressed in all control and treatment sam-
ples and that its expression was regulated differentially by the various PPAR
receptor subtype agonists tested. This novel nding that UCP-2 is expressed
in glioma cells, and that its expression is regulated by PPAR agonists, sug-
gests a potential mechanism for the cytotoxic effects of glitazones that have
been previously reported, and describes a mechanism which could possibly
be manipulated as a potential therapeutic avenue in the future.
57. GLYCOLYTIC GLIOMA CELLS SHOWED SENSITIVITY
TO INHIBITION BY A MYRISTOYLATED PEPTIDE DERIVED
FROM KEY PHOSPHORYLATION SITES ON GSK-3ALPHA/
BETA
M.E. Beckner,
1
F. Burovic,
2
and I.F. Pollack
2
;
1
Pathology;
2
Neurological
Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
U87 malignant glioma cells exhibit impressive migration in vitro. Pre-
viously, we demonstrated sensitivity of phosphatase and tensin homolog
(PTEN)-mutated U87 cell migration to restoration of PTENs constitutive
negative regulation via a wild-type construct (kind gift of R. Abounader
and J. Laterra, Johns Hopkins Univ.), and inhibition of phosphotidylinosi-
tol 3 kinase (PI3K) via wortmannin (Beckner et al., Proc. Am. Assoc. Can-
cer Res. 45, 694, 2004). In addition to regulatory effects of the upper acti-
vated PI3K/Akt pathway on the cytoskeleton, we propose that additional
activation of migration occurs downstream via activated glycogen synthase
(GS). Akt, Wnt, and other pathways converge at glycogen synthase kinase-3
(GSK-3). By phosphorylating and thus inactivating GSK-3, its constitutive
inhibitory phosphorylations of GS are suppressed. Activation of GS was
shown by decreased phosphorylation at S640 and increased phosphoryla-
tion of GSK-3alpha and Akt1 at S21 and T308, respectively, in hypoxia.
Activation of GS also occurred in U87 pseudopodia. Activated GS aids in
the removal of lactic acid in glycolytic, gluconeogenic astrocytic cells (Drin-
gen et al., Brain Res. 623, 208; Biol. Chem. Hoppe Seyler 374, 343, 1993;
Hevor, Biochemie 76, 111, 1994; Bernard-Helary K. et al., Glia 37, 379,
2002). U87 proteomics revealed a predominance of glycolytic enzymes with
increased amounts in pseudopodia. HGF, Met, actin, and other proteins
were also increased (M.E. Beckner et al., Lab Invest., in press). Intracellular
lactate removal should aid migration of invasive glioma cells in vivo, espe-
cially hypoxic pseudopodial extension. To block phosphorylation of GSK-
3, a myristoylated (Mys) peptide, Mys-CGPKGPGRRGRRRTSSFAEG,
was generated to mimic GSK-3 alpha and betas phosphorylation sites, S21
and S9, respectively, and a negative control, Mys-GRRGRRRPGCEKSPS-
GFTGA, was made. The mimics sequence was published for GSK-3 Fusion
Protein (Product #9278, Cell Signaling, Inc., Beverly, MA). The Mys-GSK-
3 peptide induced a biphasic effect, including inhibition of normoxic and
hypoxic U87 cell migration at concentrations 3-fold less than the control,
with a differential effect on trypan blue exclusion also noted. Blocking
phosphorylation of GSK-3 at cell membranes, especially pseudopodia, may
suppress invasion. This research was supported by The Nick Eric Wichman
Foundation and The Pittsburgh Foundations Walter L. Copeland Fund for
Cranial Research.
58. NOTCH2 INTRODUCES CBF1-INDEPENDENT PATHWAY
IN GLIOBLASTOMA: EVIDENCE OF NOVEL NOTCH SIGNAL
ELEMENT
M. Noha,
1
D. Yoshida,
2
D. Morimoto,
2
T. Asakura,
2
and A. Teramoto
2
;
1
Teikyo University School of Medicine, Department of Neurosurgery,
Kawasaki;
2
Nippon Medical School, Department of Neurosurgery,
Tokyo; JAPAN
Notch signaling is well known to play a crucial role in cell fate deci-
sions during development. Furthermore, in humans, four different subtypes
of Notch have already been recognized. In the past decade, many reports
have implicated the notch protein in tumorigenesis, specically because
of overexpression of the Notch intracellular domains as the constitutive
active form of Notch. However, the function of this truncated Notch is still
not clear, and a few reports have described the correlation between each
subtype of Notch and tumorigenesis. We previously reported that Notch
signaling is associated with the evasion of apoptosis of glioblastoma. In
this report, we focus on Notch1 and Notch2 expression in glioblastoma,
and we discuss the function of Notch signaling in this tumor. Glioblastoma
cell lines U373MG, U87MG, U251MG, A172, and T98G were investigated
for expression of Notch1 and Notch2 protein and mRNA by using Western
blot and RT-PCR, respectively. The intracellular distribution of these two
Notch proteins was then revealed individually by immunocytochemistry
using specic antibody to Notch1 and Notch2. Downstream of Notch sig-
naling, truncated Notch protein binds CBF1 and expresses HES1. Such
protein-to-protein interaction was also conrmed in glioblastoma cell lines
by immunoprecipitation. Finally, by using specic siRNA, Notch1 and
Notch2 expression was silenced, and subsequently, mRNA expression of
HES1, as the target gene of Notch signaling, was measured by real-time
PCR. All cell lines expressed Notch1 and Notch2 and showed a tendency
to express notch2 dominantly. In addition, intracellular Notch expressed
strongly, which indicates that Notch signaling was well activated in glio-
blastoma as well as in carcinoma of other organs. Immunocytochemistry,
immunoprecipitate, and gene silencing by siRNA more clearly differenti-
ated between Notch1 and Notch2 attributes in glioblastoma. A number of
Notch2 proteins distributed in nuclei and Notch1 in the perinuclei area of
cytoplasm. Furthermore, Notch2 no longer bound CBF1 and expression
of HES1 was not affected, which suggests that Notch2 might introduce
novel CBF1-independent pathway in glioblastoma cell lines. Notch2 signal-
ing might play an unexpected role in glioblastoma, through novel unidenti-
ed Notch pathway components.
59. CYTOPLASMIC TRANSLOCATION OF PTEN TUMOR
SUPPRESSOR IS MEDIATED BY PI3K/AKT/MTOR/P70S6K
SIGNALING CASCADES
J. Liu
1
, K. Aldape
2
, Z. Mao
1
, and W. Yung
1
;
1
Neuro-Oncology and
2
Pathology, UT M.D. Anderson Cancer Center, Houston, Texas, USA
The PTEN tumor suppressor gene is commonly deleted or mutated
in a large number of advanced tumors including GBMs. PTEN functions
primarily as a phosphoinositide phosphatase that specically antagonizes
PI3K-mediated signaling pathways. PTEN is preferentially expressed in
the nucleus of differentiated or resting cells, and increased nuclear PTEN
expression is associated with G
0
/G
1
. However, the regulation of PTENs
nuclear-cytoplasmic shuttling remains poorly understood. In this report,
we used mouse astrocytes and NIH3T3 cells to study the molecular mecha-
nisms involved in regulation of PTEN nuclear export. In agreement with
other reports, we show that PTEN is preferentially localized in the nucleus
during G
0
/G
1
phase and is exported into the cytoplasm during G
1
/S transi-
tion. We further demonstrate that dominant-negative mutants for Akt (Akt-
AAA) and for p70S6K (K113R) as well as inhibitors for PI3K (LY294002),
PDK1 (Staurosporine), mTOR (RAD-001), and sodium salicylate (for
p70S6K), but not for MEK (PD98059), suppress the nuclear export of
PTEN protein. Conversely, constitutively active AKT mutant (AKT-DD)
promotes PTENs cytoplasmic translocation. In addition, we also dem-
onstrate that PTEN interacts with p70S6K in vivo and in vitro. Taken
together, these ndings strongly suggest that PI3K/AKT/mTOR/p70S6K
signaling cascades, p70S6K in particular, are pivotal in regulating PTENs
subcellular localization. This scenario is reminiscent of yin and yang
reciprocal regulation between PI3K and PTEN during the course of cell
cycle progression. Interestingly, our immunohistochemistry results show
that PTEN is predominantly expressed in the cytoplasm of GBM tumors
that positively correlates with the phosphorylation of S6. Furthermore, we
also observe preferentially cytoplasmic localization of PTEN in a GBM
cell line, LN229, which is presumably due to the constitutive activation
of p70S6K that can be blocked by sodium salicylate but not by RAD-001.
Our long-term goal is to establish the relationship between PTENs subcel-
lular localization and the status of activation of PI3K downstream effectors,
such as AKT, mTOR, or p70S6K in order to improve molecular diagnosis,
prognosis, and individualized therapy for GBMs.
60. THE NOTCH SIGNALING PATHWAY AND GROUCHO/
TLE CO-REPRESSORS IN MENINGIOMA PATHOGENESIS
I. Cuevas, G. Baia, A. Jun, P. Costello, J. Bollen, A. McDermott, and A.
Lal; Brain Tumor Research Center, Neurological Surgery, University of
California San Francisco, San Francisco, California, USA
Meningiomas constitute the second most common central nervous
system tumor. Atypical and malignant meningiomas are associated with
a poor clinical outcome and higher rates of recurrence when compared to
benign meningiomas. Relatively little is known about the molecular events
important in the pathogenesis and malignant progression of meningiomas.
To determine the molecular changes associated with meningiomas, we used
serial analysis of gene expression (SAGE). We focused our initial analysis
on the 165 genes that are induced in high-grade meningiomas because this
population is expected to contain components of activated signal transduc-
tion pathways. A novel nding from this screen is the induction of three
downstream components of the Notch signaling pathway: the transcrip-
tion factor, Hairy and Enhancer of Split 1 (HES1), and two members of
the Groucho/Transducin-like enhancer of split (Gro/TLE) family of co-
repressors, TLE2 and TLE3. Gro/TLE co-repressors interact and modulate
the activity of a wide range of transcriptional regulatory systems, one of
which is HES1. The SAGE results were validated by performing quantita-
tive PCR on a larger, independent set of meningiomas. We conrm that
HES1 transcript levels are induced in meningiomas of all three grades while
induction of TLE2 and TLE3 is specic to high-grade meningiomas. In
particular, overexpression of TLE3 occurs in 50% of malignant menin-
giomas, with the amplitude of induction ranging from 4-fold to 65-fold.
Immunohistochemistry revealed that TLE3 is correctly localized to the
nucleus, where it is supposed to function as a transcriptional co-repres-
sor. We also nd induction of other components of the Notch signaling
pathway like NOTCH2 and JAGGED1 in a subset of meningiomas. The
above results lead us to hypothesize that Gro/TLEs are important for the
malignant progression of meningiomas and that one of the mechanisms by
which this occurs is by modulation of the Notch signaling pathway. We are
currently overexpressing and silencing components of the Notch signaling
pathway and TLE repressors to delineate their relevance in meningioma
growth and tumorigenesis.
61. THE IN VITRO ANTICANCER ACTIVITY OF GLIVEC
AGAINST GLIOBLASTOMA MULTIFORME IS NOT RELATED
TO PDGFRAND KIT EXPRESSION
S. Spiegl-Kreinecker,
1
C. Pirker,
2
R. Silye,
3
J. Buchroithner,
1
J. Pichler,
4

J. Fischer,
1
M. Micksche,
2
and W. Berger
2
;
1
Department of
Neurosurgery, Wagner Jauregg Hospital, Linz;
2
Clinics of Internal
Medicine I, Institute of Cancer Research, Vienna;
3
Institute of Pathology,
Wagner Jauregg Hospital, Linz;
4
Department of Internal Medicine,
Wagner Jauregg Hospital, Linz; Austria
Overexpression of the tyrosin kinase receptors (RTKs) PDGFRa/b and
c-Kit is a common event in glioblastomas, therefore representing obvious
targets for the small molecule inhibitor Glivec. Moreover, the PDGFRa
gene is amplied in a small subgroup of glioblastoma patients. The aim
of our study was to compare the expression of PDGFR isoforms a and b
as well as c-Kit with the therapeutic efcacy of Glivec in glioblastoma pri-
mary cell cultures. Western blot was performed to detect respective protein
levels of PDGFRa/b and c-Kit in 43 primary cell cultures from astrocytic
brain tumor surgery specimens. Tumor samples were obtained during sur-
gery and histologically veried according to WHO criteria as glioblastoma
multiforme. Immunohistochemical expression of PDGFRa and c-Kit was
determined in selected parafn sections (N 5). Sensitivity against Glivec
(1050 M) was analyzed by MTT tests. Chromosomal aberrations were
studied by means of comparative genomic hybridization (CGH) using DNA
isolated from cell cultures as well as corresponding tumor sections. Of 43
primary cell cultures, 30 (70%) expressed detectable levels of PDGFRa;
34/43 (79%) displayed PDGFRb protein expression, whereas 25/43 (58%)
showed expression of both receptor isoforms. Expression of c-Kit was
detectable in 17/43 (40%) of the analyzed cell cultures. In one highly PDG-
FRa-overexpressing cell culture, high-level amplication of the respective
gene at chromosome 4q12 was detected by CGH. This expression level was
well in accordance with response to Glivec. Four of ve tumor sections
displayed heterogenous PDGFRa immunostaining. Staining intensity dif-
fered between and within samples and was high in giant tumor cells and
endothelial cells. Widespread but weak staining could be observed in bril-
lary tumor cells. In one case, no immunoreactivity was detectable. PDGFRa
immunostaining of tumor sections corresponded well with respective cell
cultures. This was not the case for c-Kit. Sensitivity against Glivec could be
observed in 19/21 investigated cell cultures, with IC
50
ranging between 19
and 42 M. Two of 21 were resistant against Glivec despite high expression
level of PDGFRa and c-Kit, respectively. Sensitivity against Glivec did not
correlate with expression of any of the investigated RTKs. Summing up,
our data demonstrate frequent expression of PDGFRa/b and/or c-Kit in
glioblastoma cells. However, the in vitro chemosensitivity against Glivec
is not related to expression levels of these targeted RTKs. The relationship
of PDGFRa gene amplication in a small subgroup of glioma patients and
Glivec sensitivity should be further investigated, since a glioblastoma cell
culture with PDGFRa gene amplication was sensitive against Glivec in
our in vitro experiments.
62. THE PTEN TUMOR SUPPRESSOR GENE SHIFTS
CELLULAR RESPONSES TO TRANSFORMING GROWTH
FACTOR-BETA TOWARDS TUMOR SUPPRESSION IN
MALIGNANT GLIOMAS
A. Hjelmeland,
1
M. Hjelmeland,
1
C. Kontos,
2
D. Bigner,
3
X. Wang,
1

and J. Rich
2
;
1
Surgery;
2
Medicine;
3
Pathology;
4
Pharmacology & Cancer
Biology, Duke University, Durham, North Carolina, USA
Transforming growth factor- (TGF) is a multifunctional cytokine
commonly expressed by malignant gliomas that regulates a diverse set of
biological activities, including proliferation, apoptosis, differentiation,
motility, extracellular matrix deposition, and angiogenesis. Although glial
cells are growth inhibited by TGF, glioma cell lines are resistant to TGF-
mediated growth inhibition yet retain responsiveness to the effects of TGF
on the neoplastic phenotypesecretion of angiogenic factors, induction of
invasion, and immune escape. The molecular mechanisms through which
TGF shifts from a tumor suppressor to a tumor enhancer in advanced
cancers are poorly understood. Recent work suggests that the phosphati-
dyl 3-OH inositol kinase (PI3K) pathway interacts with TGF signaling
at multiple levels. We therefore sought to determine the functional signi-
cance of PTEN expression on TGF-mediated transcription. Restoration of
wild-type PTEN into a PTEN null glioma cell line inhibited TGF-induced
transcription in a PTEN concentration-dependent fashion, whereas mutant
PTEN lacking both protein and lipid phosphatase activity did not have a
similar effect. In a cell line with wild-type PTEN expression, stable knock-
down of PTEN expression with short hairpin RNA (shRNA) increased
TGF transcriptional activation or inhibition in multiple TGF responsive
promoters. Results with these luciferase reporters suggest that PTEN func-
tions through a mechanism distinct from other PI3K pathway components
previously shown to interact with SMADs. To elucidate the mechanism
by which PTEN inhibits TGF transcription, we examined TGF-induced
phosphorylation of key intracellular mediators (the SMADs) with PTEN
expression. C-terminal SMAD phosphorylation was moderately decreased
with wild-type PTEN expression but not mutant PTEN, including a mutant
that retains protein phosphatase activity. Reconstitution of wild type but
not phosphatase dead mutant PTEN into PTEN null glioma cell lines re-
established growth inhibition in response to TGF. In keeping with the
biphasic nature of TGF signaling, PTEN reconstitution blocked inva-
sion through an articial matrix induced by TGF. Reciprocally, inhibit-
ing PTEN expression in a PTEN wild type cell line through expression
of shRNA directed against PTEN inhibited TGF-induced motility. Thus,
loss of PTEN expression may promote cellular responses to TGF involved
in tumor progression. Reintroduction of PTEN may shift TGF cellular
responses towards a tumor suppressive phenotype by restoring sensitivity
of glioma cell lines to TGF-mediated growth inhibition while blocking
TGF-mediated invasion. This work was also supported by NIH grants
NS047409. J.N.R. is a Damon Runyon-Lilly Clinical Investigator and a
Sidney Kimmel Cancer Foundation Scholar.
63. RNAi-MEDIATED SIMULTANEOUS DOWNREGULATION
OFUPAR AND uPA RESULTS IN THE DOWNREGULATION
OF THE REGULATORY ASSOCIATED PROTEIN OF mTOR
(RAPTOR), A COLLAPSE IN MITOCHONDRIAL DY, AND
THE INDUCTION OF PRO-APOPTOTIC GENES IN SNB19
HUMAN GLIOMA CELLS
C. Gondi, S. Kondraganti, W.C. Olivero, D. Dinh, M. Gujrati, and J.S.
Rao; Department of Biomedical and Therapeutic Sciences, UIC College
of Medicine at Peoria, Peoria, Illinois, USA
Our previous results have demonstrated the ability of a plasmid-based
RNAi system to silence uPAR and uPA using a bicistronic construct. Uroki-
nase plasminogen activator (uPA) and its receptor (uPAR) are overexpressed
during glioma cell invasion and progression. Pro-uPA, when bound to its
receptor uPAR, is activated to uPA, which in turn activates plasminogen to
plasmin. The binding of uPA to uPAR initiates a cascade of signaling events
involving integrins mediated by MEK and PI3-K. SNB19 cells were trans-
fected with plasmid-expressing RNAi targeting uPAR (pUR), uPA (pUP),
uPAR-uPA simultaneously (pU2), empty vector (EV), or scrambled vector
(SV). Western blot analysis of RAPTOR (regulatory associated protein of
mTOR) showed a marked decrease in expression levels when transfected
with plasmids expressing either pUP or pU2. In contrast, cells transfected
with pUR did not exhibit a decrease in RAPTOR levels, which indicates
that uPA levels are integral in maintaining the activity of mTOR. In addi-
tion, Ki67 levels decreased in pUP- and pU2-transfected cells. However,
downregulation of uPAR alone did not result in a decrease of Ki67 levels,
further indicating the involvement of uPA with proteins other than its tra-
ditional receptor. Mitochondrial transition event was measured by using
uorescent cation dye 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzamid-
azolocarbocyanin iodide, where red uorescence indicates DY collapse. The
simultaneous downregulation of uPAR and uPA resulted in a decrease of red
orescence, whereas the downregulation of either uPAR or uPA alone did
not. These data strongly suggest the involvement of the uPA uPAR complex
in mitochondrial DY maintenance and collapse. The simultaneous down-
regulation of uPAR and uPA also induced caspase 8 activation accompanied
by cytochrome c release, thereby indicating the initiation of apoptosis. Of
further interest is that, in pU2-transfected cells, PARP levels were elevated
and cleaved PARP levels were decreased 72 h after transfection, but 120 h
after transfection, PARP cleavage, an indicator of apoptotic progression,
was observed. In conclusion, the simultaneous downregulation of uPAR
and uPA inhibited cap-dependent mRNA translation via downregulation
of RAPTOR, initiated apoptosis by mediating the collapse of mitochondrial
DY, and induced caspase 8 and PARP cleavage. These results clearly suggest
that the simultaneous targeting of uPAR and uPA has potential for cancer
gene therapy.
64. OVEREXPRESSION OF MARCKS IN EGFRvIII-
EXPRESSING GLIOBLASTOMA MULTIFORME
J. Micallef,
1
J. Caldwell,
1
M. Moran,
1
and A. Guha
1,2
;
1
Neurosurgery
Division, Toronto Western Hospital, Toronto, Ontario;
2
HSC,
Neurosurgery Division, Labatts Brain Tumor Centre, Toronto, Ontario;
Canada
The most common gain of function mutation in malignant astrocy-
tomas (GBMs) is amplication or overexpression of the wild-type (wt)
and mutated epidermal growth factor receptors (EGFRs), the most com-
mon of which is EGFRvIII. The differential signaling pathways utilized
by EGFRvIII vs. wt-EGFR, contributing to its more aggressive behavior,
is not known. ICAT, a mass specbased analytical technique to evaluate
differential protein proles, demonstrated that GBM explant xenografts
harboring EGFRvIII expressed much higher levels of MARCKS, as com-
pared to GBMs, which express only wt-EGFR. MARCKS has been previ-
ously implicated in tumor invasion and breast cancers, but not in gliomas
or EGFR signaling. MARCKS overexpression in EGFRvIII GBMs was
veried by Western immunoblot analysis on a larger panel of GBM cell
lines with Tet-Off expression of EGFRvIII or wtEGFR, GBM xenografts
with EGFRvIII and GBM operative specimens with EGFRvIII. Differ-
ences at the level of the proteome, evaluated by proteomic-based techniques
such as ICAT, allow us to understand biological and clinical similarities or
differences between subtypes of human diseases, such as GBMs. Current
work involves assessing the functional role of MARCKS overexpression
in GBMs. We will discuss our experiments with siRNA downregulation
of MARCKS in GBMs expressing EGFRvIII and MARCKS overexpres-
sion in GBMs expressing only wt-EGFR. If MARCKS contributes to the
aggressive biology of EGFRvIII expressing GBMs, it may be an additional
biological target.
65. OVEREXPRESSION OF INDUCIBLE NITRIC OXIDE
SYNTHASE AND MAINTENANCE OF MALIGNANT
PHENOTYPE IN MENINGIOMAS
I. McCutcheon,
1
K. Bian,
2
J. Li,
1
and F. Murad
2
;
1
M.D. Anderson Cancer
Center, Neurosurgery, and
2
Integrative Biology and Pharmacology,
Houston Medical School, The University of Texas, Houston, Texas, USA
Because metastatic activity, host defense mechanisms, and level of dif-
ferentiation seem to be correlated to iNOS expression, a role for iNOS
has been proposed in the onset and progression of malignant diseases.
Under physiological condition, NO acts as an intracellular secondary
messenger and provides an efcient system for cellular regulation, interac-
tion, and defense. The expression of 3 isoforms of NOS in human gliomas
and in peritumoral areas has been analyzed by several groups. Although
the induction of iNOS has been noted in human meningioma (Bakshi et
al., 1998; Broholm et al., 2003; Ellie et al., 1995; Hara et al., 1996), a
clear correlation between meningiomas and iNOS expression has not
been established. Human meningiomas of benign, atypical, and anaplas-
tic/malignant grade (n 10) were excised and primary explant cultures
obtained. Histopathological conrmation of grade was obtained. By using
Western blot, expression of inducible nitric oxide synthase (iNOS) was
demonstrated for cultured cells and intact tissue fragments. Integrity of
downstream NO signaling pathways was tested by exposing cultured cells
at early passage to NO donor compounds. The downstream actions of NO
take two forms, (1) cGMP-dependent; and (2) cGMP-independent, which
are mediated by reactive nitrogen species produced by the interaction of
NO with O
2
or with superoxide radicals (O
2
.
). The rst was tested by

measuring levels of guanyl cyclase and of cGMP, and the second by assay
of nitrotyrosine formation, in NO-donor-stimulated cells of each grade.
A clear correlation was shown between iNOS expression and the degree
of tumor malignancy, with lowest expression seen in benign tumors and
highest seen in those of anaplastic grade. In malignant meningioma cells
both membrane-bound and soluble guanylyl cyclase (sGC) were un-regulat-
able, and the cGMP levels were very low despite signicantly high levels of
iNOS expression. In contrast, in benign meningioma cells, sGC responded
to NO normally, and a marked increase of cGMP was observed upon NO
donor stimulation. Thus, the downstream pathways of NO signaling in
malignant meningioma appear to be shut down. In additional experiments,
a signicant increase in nitrotyrosine formation (a biomarker for cGMP-
independent NO action) was detected in malignant cells. We conclude that
iNOS expression correlates with degree of malignancy in meningiomas.
However, in anaplastic meningiomas, cGMP-dependent signaling path-
ways are inactive, but cGMP-independent pathways are not. These data
suggest that in malignant meningioma cells, iNOS overexpression may
contribute to a survival advantage mediated by protein tyrosine nitration.
66. MOLECULAR GENETIC STUDY FOR
HEMANGIOBLASTOMAS AND FUNCTIONS OF VHL GENE
H. Kanno,
1
A. Kubo,
1
T. Mimura,
1
Y. Tanaka,
1
and I. Yamamoto
1
;
1
Yokohama City University Graduate School of Medicine, Neurosurgery,
Yokohama, Japan
Molecular genetic analysis for germline or somatic mutation of von
Hippel-Lindau (VHL) gene in central nervous system (CNS) hemangio-
blastomas (HBs) with and without VHL disease contributes to surgical
treatment and follow-up. Forty-nine patients bearing HBs (sporadic 33,
VHL 16) underwent surgery and genetic diagnosis for germline and somatic
VHL mutations. Locations of the tumors were cerebellum, 39; brain stem,
3; and spinal cord, 10. Fourteen of 16 VHL cases had multiple tumors while
3 of 32 sporadic cases had one tumor. Twelve sporadic HBs showed somatic
mutations (missense 6, truncation-type 6) but not germline mutations. In
addition, 24 sporadic HBs showed loss of heterozygosity (LOH) on 3p, in
which the VHL gene is located. These results suggested that the inactiva-
tion of VHL genes on both alleles was a cause of genesis in the majority of
sporadic hemangioblastomas and that the VHL gene functioned as a tumor
suppressor gene. Eleven of 15 VHL cases showed VHL germline mutations
(missense 8, truncation-type 4). Patients with truncation-type VHL germ-
line mutation were more frequently associated with renal cell cancer (RCC).
Causes of death were postoperative complications in 2 sporadic patients
and tumor development in 2 VHL patients. Clinically ambiguous cases,
whether sporadic or VHL, should be analyzed for VHL germline muta-
tion. It might be recommended that HBs with VHL should be surgically
treated if symptomatic, while asymptomatic small ones should be observed
or treated with radiosurgery. Functions of the VHL gene include not only
tumor suppression in HB as the above but also neuronal differentiation,
which we demonstrated. Herein we show neuronal regeneration with donor
of VHL-gene or peptide transferred stem cells (neural stem cell, bone mar-
row stromal cell, skin stem cell, ES cell). Transplantation with VHL-gene
transferred stem cells dramatically improved symptoms of neuronal dis-
ease model rats (Parkinson, cerebral infarction, spinal injury), and they
functioned as neurons in the brain. It was suggested that neuronal differ-
entiation by VHL protein was related to ubiquitination and resolution of
Notch under normoxia but not under hypoxia. In addition, synthetic VHL
oligopeptide (elongin-binding site at a-domain) showed induction potential
for neuronal differentiation equal to transduction with viral vector. In the
future, neuronal regeneration with VHL gene or peptide would be useful
for the clinical level.
67. LIGAND-INDEPENDENT ACTIVATION OF THE
EGFRvIII: A NATURALLY OCCURRING MUTATION OF THE
EGFR COMMONLY EXPRESSED IN GLIOMA
T. Johns, A.A. Vitali, R.M. Perera, S. Vernes, and A.M. Scott; Ludwig
Institute for Cancer Research, Oncogenic Signalling, Heidelberg,
Australia
Mutations of the epidermal growth factor receptor (EGFR) gene are
found at a relatively high frequency in glioma, with the most common being
the de2-7 EGFR (or EGFRvIII). This mutation arises from an in-frame dele-
tion of exons 27, which removes 267 amino acids from the extracellular
domain of the receptor. Despite being unable to bind ligand, the de2-7
EGFR is constitutively active at a low level. Transfection of human glioma
cells with the de2-7 EGFR has little effect in vitro, but when grown as tumor
xenografts this mutated receptor imparts a dramatic growth advantage. We
have now mapped the phosphorylation pattern of de2-7 EGFR, both in vivo
and in vitro, using a panel of antibodies unique to the different phosphory-
lated tyrosine residues. Phosphorylation of de2-7 EGFR was detected con-
stitutively at all tyrosine sites surveyed both in vitro and in vivo, including
tyrosine 845, a known target in the wild-type EGFR for src kinase. There
was a substantial upregulation of phosphorylation at every tyrosine residue
of the de2-7 EGFR when cells were grown in vivo compared to the receptor
isolated from cells cultured in vitro. Upregulation of phosphorylation could
be mimicked in vitro by the addition of specic components of the ECM
such as collagen via an integrin-dependent mechanism. Since this increase
in in vivo phosphorylation enhances de2-7 EGFR signaling, this observa-
tion explains why the growth enhancement mediated by de2-7 EGFR is
largely restricted to the in vivo environment. In a second set of experiments
we analyzed the interaction between EGFRvIII and ErbB2. Co-expression
of these proteins in NR6 cells, a mouse broblast line devoid of ErbB fam-
ily members, dramatically enhanced in vivo tumorigenicity of these cells
compared to cells expressing either protein alone. Detailed analysis of these
xenografts demonstrated that EGFRvIII could heterodimerize and trans-
phosphorylate the ErbB2. Since both EGFRvIII and ErbB2 are commonly
expressed at gliomas, this data suggests that the co-expression of these two
proteins may enhance glioma tumorigenicity.
68. LONG-TERM SURVIVAL IN PATIENTS WITH
GLIOBLASTOMA MULTIFORME TREATED IN PHASE 2
STUDIES WITH ANP
R.A. Weaver, S.R. Burzynski, T.J. Janicki, B. Burzynski, G. Jurida, and
B. Szymkowski; Burzynski Clinic, Houston, Texas, USA
The purpose of this study was to determine the frequency of long-term
survivals in patients with glioblastoma multiforme (GBM) treated in FDA
and Institutional Review Board monitored phase 2 studies with ANP (anti-
neoplastons A10 and AS2-1). One hundred seventy-three patients with
GBM who could be evaluated were accrued to FDA-monitored phase 2
trials. Seventy-nine patients were admitted to the study protocols (SP), and
an additional group of 94 patients were treated under special exception (SE)
because of low Karnofsky performance status (KPS), below 60. Ninety-
eight percent of patients failed prior surgery, radiation therapy, and/or che-
motherapy. ANP was given intravenously daily in escalating doses. The
median duration of ANP administration was 4 months for SP and 3 months
for SE, and the average dosage of A10 was 6.18 (SP) and 6.92 (SE) and of
AS2-1 was 0.26 (SP) and 0.25 (SE) g/kg/d. Responses were assessed by
gadolinium-enhanced MRIs and PET scans (as necessary). Long-term sur-
vival was dened as patients surviving 3 years after initial diagnosis. There
was 15.5% long-term survival in the SP group and 7.1% in the SE group.
The maximum survival in the SP group was more than 12 years and in the
SE group was more than 10 years. Survival was signicantly reduced in the
SE group, which consisted of patients with lower KPS. The data indicate
that more than 15% of evaluable patients with GBM treated with ANP in
phase 2 studies were long-term survivors. The results are signicantly worse
in a group of patients with lower KPS, but compare favorably with radiation
therapy and chemotherapy.
69. RESPONSES OF THE ADULT MAMMALIAN CENTRAL
NERVOUS SYSTEM TO EXPERIMENTAL INTRACRANIAL
GLIOMA
M. Barish,
1
J. Najbaur,
2
E. Garcia,
2
M. Metz,
2
and K. Aboody
2
;
1
Division of Neurosciences, Beckman Research Institute and
2
Division of
Hematology and HCT, City of Hope National Medical Center, Duarte,
California, USA
In the brain, tumor cells and normal cells are the interacting elements of
a two-part system connected by competition for physical space, extracellular
matrix components, and secreted soluble factors. Our goal is to understand
how normal brain cells respond to, and feed back onto, tumor cells. In the
present experiments, U251 human glioma cells were injected into cortices of
adult nude mice to induce experimental glioma. Brains were examined over
time when tumors were 0.5 to 4 mm in diameter and occupying an increas-
ing portion of the frontal area. Markers of cell type and status are examined
by immunouorescence in horizontal sections. Nestin immunoreactivity (ir)
was detected in tumor and host cells and separated using species-specic
antibodies (Chemicon; MAB353 for mouse, MAB5326 for human). On the
ipsilateral side, nestin ir was observed in host cells along both lateral and
medial ventricular walls, in cells positioned between the tumor mass and
the pia, in cells dispersed around the tumor and just inside its edge, and in
the corpus callosum and adjacent tumor. Some nestin-immunoreactive
(nestin-ir) cells, especially those in the parenchyma lateral to the ventricle
and inside the tumor mass, had the morphology of neural progenitor cells
(NPCs). Other nestin-ir cells, particularly those interposed between the
tumor and the pia, had the brous morphology of reactive astrocytes, and
some of these cells were also GFAP-ir. The presence of proliferating cells
was probed using antibody to PCNA (Chemicon MAB424). In addition
to the tumor cells, PCNA-ir was observed in subpopulations of nestin-ir
presumptive NPCs and in nestin-ir presumptive reactive astrocytes, as well
as in unidentied non-nestin-ir cells dispersed lateral to the ventricle, in
the corpus callosum, in the mbria, and in the dentate gyrus. Our results
suggest that the experimental glioma induced appearance of proliferating
nestin-ir NPCs, nestin-ir reactive astrocytes, and other cells whose identity
has not yet been determined. The position of the reactive astrocytes sug-
gests that mechanical deformation may play an inductive role. NPCs near
the ventricle and medial to the tumor mass may be responding to presently
unidentied factors emerging from the tumor and/or from adjacent host
cells. In neither case did it appear that the tumor environment was highly
mitogenic for host cells, which suggests that host cells were not major con-
tributors to the tumor burden.
70. TARGETED THERAPY WITH ANP IN CHILDREN LESS
THAN 4 YEARS OLD WITH INOPERABLE BRAIN STEM
GLIOMAS
S.R. Burzynski, R.A. Weaver, T.J. Janicki, B. Burzynski, and G. Jurida;
Burzynski Clinic, Houston, Texas, USA
The purpose of the study is to evaluate the outcome of patients less
than 4 years old with intrinsic diffuse brain stem gliomas (BSG) treated
with ANP (antineoplastons A10 and AS2-1) in two FDA and Institutional
Review Board monitored phase 2 trials. A total of 10 assessable patients
who were less than 4 years old were among 65 participants of phase 2 trials
(study and special exception patients): 2 with anaplastic astrocytomas, 1
with pilocytic astrocytoma, and 7 who had no biopsy because of a danger-
ous tumor location. Age ranged from 3 months to 3 years. Three patients
failed prior radiation and chemotherapy, 1 had stable disease after radia-
tion, 2 had tumor resection, and 4 were not treated prior to ANP. ANP
was given intravenously daily through a subclavian venous catheter and a
double channel infusion pump. The median duration of ANP administra-
tion was 9 months, and the average dosage of A10 was 12.16 g/kg/day
and of AS2-1 was 0.41 g/kg/day. Responses were assessed by gadolinium-
enhanced MRIs and conrmed by PET scans in some cases. The overall
survival at 2 years was 50% and at 5 years 20%, and the maximum survival
is 6 years. Median progression-free survival was 2 years and 2 months.
Complete response was achieved in 30%, stable disease in 40%, and pro-
gressive disease in 30%. Serious toxicities included reversible anemia and
hypokalemia. There were no chronic toxicities. ANP targets the AKT2,
RAS, p53, and p21 pathways, and its administration results in substantial
survival and response rates in a small group of young children who do not
have a favorable prognosis for standard therapy.
71. INHIBITION OF INSULIN-LIKE GROWTH FACTOR I
RECEPTOR SIGNALING INCREASES CHEMOSENSITIVITY
OF PEDIATRIC CNS ATYPICAL TERATOID/RHABDOID
TUMOR CELLS
T. Shalaby, J. Dcunia, and M. Grotzer; University Childrens Hospital,
Oncology, Zurich, Switzerland
Central nervous system (CNS) atypical teratoid/rhabdoid tumors (ATT/
RhT) are among the pediatric malignant tumors with the worst prognosis
and fatal outcome. To date there are no explanations for their remarkable
resistance to cytostatic drugs and radiotherapy. Insulin-like growth factor I
receptor (IGF-IR) protects cancer cells from apoptosis induced by a variety
of anticancer drugs and radiation, but when impaired by inhibitors, tumor
cells undergo massive apoptosis, resulting in an inhibition of tumorigenesis
and metastases in experimental animal models. IGF-IR was found to be
clearly overexpressed in ATT/RhT compared to near normal brain samples
and to other pediatric CNS neoplasms as conrmed by Western blotting
and immunohistochemistry. Moreover, we found IGF-I and IGF-II mRNA
in an ATT/RhT cell line indicating the presence of an autocrine/paracrine
IGF-I/II/IGF-IR loop in ATT/RhT. Human BT-12 and BT-16 ATT/RhT
cells were treated with IGF-IR antisense or scrambled control oligonucle-
otides for different time periods. Antisense treatment of ATT/RhT cells for
48 h resulted in signicant downregulation of IGF-IR mRNA and IGF-
IR protein expression, both in BT-12 and in BT-16 cells. IGF-IR antisense
treatment resulted in inhibition of cellular proliferation and induction of
apoptosis. Moreover, chemosensitivity of ATT/RhT cells to doxorubicin
and cisplatin was found to be signicantly increased upon treatment with
IGF-IR antisense oligonucleotides. This suggests the presence of an auto-
crine/paracrine IGF-I/II/IGF-IR loop in ATT/RhT that may be responsible
for the low susceptibility of ATT/RhT cells to undergo apoptosis. Inhibition
of IGF-IR represents a novel therapeutic strategy in childhood CNS ATT/
RhT that warrants further investigation.
72. HYPOXIA SENSITIZES HUMAN MALIGNANT GLIOMA
CELLS TOWARD CD95L-INDUCED CELL DEATH
J. Steinbach,
1
H. Wolburg,
2
A. Klumpp,
1
and M. Weller
1
;
1
Abt.
Allgemeine Neurologie and
2
Institute of Pathology, University of
Tbingen, Tbingen, Germany
Death ligands such as CD95 ligand (CD95L) have limited activity
against glioma cells under normoxic conditions (Weller et al., J. Clin. Invest.
94, 954, 1994). However, many glioma cell lines can be sensitized toward
death ligandinduced apoptosis by inhibition of epidermal growth factor
receptor (EGFR) (Steinbach et al., Brain Pathol. 12, 12, 2002). Hypoxia is
a critical aspect of the microenvironment of gliomas. We have established
a paradigm for the investigation of hypoxia-induced cell death in glioma
cells in vitro, which faithfully reproduces many aspects of human glioma
pathology (Steinbach et al., Cell Death Differ. 10, 823, 2003). Here, we
investigated the effect of co-exposure to acute hypoxia and CD95L in three
human malignant glioma cell lines with different susceptibility to CD95L
under normoxic conditions. Hypoxia sensitized all three cell lines toward
CD95L-induced cell death. Co-exposure resulted in apoptotic changes in
the early phase, with gradual conversion to secondary necrosis with increas-
ing length of hypoxia. The mitochondrial injury induced by hypoxia was
enhanced by co-treatment, and caspase cleavage became prominent. Inhibi-
tion of the EGFR, while sensitizing glioma cells to CD95L under normoxia,
protects glioma cells from hypoxia by reducing energy consumption (Stein-
bach et al., Cancer Res. 64,1575, 2004). However, the opposing effects of
EGFR signaling on death induced by CD95L or hypoxia were neutralized
by co-exposure to hypoxia and CD95L. Further, inhibition of protein syn-
thesis by cycloheximide also reduced glucose consumption and conferred
protection from hypoxia, but did not modulate CD95L-induced cell death
under hypoxic conditions. These results suggest that death ligands may be
useful to target hypoxic tumor cells resistant to conventional therapies or to
complement strategies aiming at the induction of tumor hypoxia.
73. CONSTITUTIVE INTEGRIN ACTIVATION BY RAP-
GTPASE ON LEUKEMIC CELLS PROMOTES PROGRESSION
OF LEPTOMENINGEAL LEUKEMIA
D. Brandsma,
1,2
L. Ulfman,
3
J. Reijneveld,
1
M. Bracke,
4
M. Taphoorn,
1

J. Zwaginga
6,7
M. Gebbink,
5
H. de Boer,
8
and E. Voest
2
;
Departments of
1
Neurology,
2
Medical Oncology,
3
Pulmonary Diseases,
4
Pharmaco-epidemiology and Pharmacotherapy, and
5
Haematology,
University Medical Center Utrecht, Utrecht;
6
Department of
Haematology, Academical Center Amsterdam, Utrecht;
7
Sanquin
Research, Amsterdam;
8
Department of Nephrology, Leiden University
Medical Center, Leiden; The Netherlands
Leptomeningeal metastases are a serious neurological complication in
cancer patients and are associated with a dismal prognosis. Tumor cells that
enter the subarachnoid space adhere to the leptomeninges and form tumor
deposits. The role of integrins in tumor cell adhesion to leptomeninges is
largely unknown. We studied the role of integrin expression and activa-
tion in the progression of leptomeningeal metastases. Therefore, we used a
suspension (L1210-S) and an adherent (L1210-A) variant of a mouse acute
lymphocytic leukemic cell line. Static adhesion levels of L1210-A cells on a
leptomeningeal cell layer were signicantly higher than for L1210-S cells.
All mice that were intrathecally injected with L1210-A cells died rapidly
because of leptomeningeal leukemia. In contrast, 45% of long-term survi-
vors were seen after intrathecal injection with L1210-S cells.
1
-,
2
- and
3
-integrins were in a constitutive active state on L1210-A cells and in a low
but inducible state on L1210-S cells, as determined by adhesion assays on
surfaces coated with
1
integrin ligand (collagen),
2
integrin ligand (ICAM-
1), and
3
integrin ligand (vitronectin). Expression levels of these integrins
were comparable in the two cell lines. The discrepancy in integrin activa-
tion state on the two cell lines is due to a difference in the activation of the
small GTPase Rap, which is involved in integrin inside-out signaling. Rap
in L1210-A cells is in a constitutive active state, whereas in the L1210-S
cells Rap is off, but inducible. Our data indicate that constitutive integrin
activation on leukemic cells promotes leptomeningeal leukemia progression
by increased adhesion to the leptomeninges via an aberrantly regulated Rap
GTPase signaling pathway.
74. INHIBITION OF THE MAMMALIAN TARGET OF
RAPAMYCIN SENSITIZES GLIOMA CELLS TO ANTICANCER
DRUGS
K. Sakurada,
1
Y. Sonoda,
1
C. Kitanaka,
2
W. Mori,
1
and T. Kayama
1
;
1
Neurosurgery;
2
Molecular Cancer Science, Yamagata University,
Yamagata, Japan
The phosphatase and tensin homolog deleted from chromosome 10
(PTEN) functions as a tumor suppressor by negatively regulating the
growth/survival signals of the phosphatidylinositol 3-kinase (PI3K)/Akt
pathway. The PI3K/Akt pathway in PTEN-decient tumors may be one
of the key targets for anticancer therapy. Nevertheless, how PI3K/Akt
pathway contributes to clinical drug resistance is unclear. The mammalian
target of rapamycin (mTOR) is a serine-threonine kinase that functions
downstream from Akt in PI3K/Akt/mTOR signaling pathway commonly
activated in GBM. In this study, we analyzed the effects of mTOR inhibitor
rapamycin on apoptosis and cytotoxicity induced by several kinds of che-
motherapeutic agents. Human malignant glioma cell lines T98G, U251MG
(PTEN-decient cells), and A172 (PTEN-wild-type cells) were used for this
study. We examined effects of the mTOR inhibitor rapamycin on apoptosis
and cytotoxicity induced by chemotherapeutic agents including antimi-
crotuble agent vincristine, a topoisomerase II inhibitor etoposide, and a
DNA cross-linking agent cisplatin (cis-diamminedichloroplatinum), and we
compared the rapamycin-induced enhancement of effects of those agents.
Among chemotherapeutic agents, vincristine has the strongest cytotoxicity
to the glioma cells. The rapamycin itself inhibited growth of glioma cells,
but did not cause apoptosis. In addition, the rapamycin could enhance the
cytotoxicity by vincristine. These ndings show that mTOR is a possible
target for therapy in patients with gliomas, and mTOR inhibition in combi-
nation with chemotherapeutic agents could be potent modalities for patient
with malignant gliomas.
75. CASPASE-8 STATUS OF EX VIVO GLIOMAS
D. Ashley, C. Riffkin, A. Muscat, and C. Hawkins; Royal Childrens
Hospital, Childrens Cancer Centre, Melbourne, Australia
We have identied that a signicant proportion of glioma patient sam-
ples carry mutations in the caspase-8 gene, some of which act as dominantly
interfering inhibitors. Malignant glioma has a dismal prognosis, partly
because of the inability of chemotherapy and radiotherapy to induce apop-
tosis in the tumor cells. Death ligands, including TRAIL/Apo2L, are attrac-
tive candidate therapeutic agents for unresponsive cancers like malignant
glioma, as they induce apoptosis via a pathway that is distinct from that
triggered by chemotherapy and irradiation. Caspase-8 is an apoptotic pro-
tease that plays a crucial role in apoptosis mediated by death ligands. Muta-
tions or downregulation of caspase-8 have been reported in neuroblastoma
and other tumor types, prompting its designation as a tumor suppressor
gene. In this study, we analyzed the status of caspase-8, and other apoptosis
pathway components, in ex vivo high-grade glioma specimens. Caspase-8
protein levels were frequently low or undetectable. We also amplied and
sequenced the caspase-8 mRNA expressed by the tumors. A signicant pro-
portion of the gliomas bore mutations, some of which acted as dominant
negative inhibitors in death ligand apoptosis assays. These data have sig-
nicant implications for our understanding of the process of gliomagenesis
and the potential use of death ligands like TRAIL for treatment of patients
with malignant glioma.
76. EXPRESSION LEVELS OF THE TRANSFORMING
GROWTH FACTOR-BETA MEDIATOR SMAD3 DETERMINES
GLIOMA PHENOTYPE IN A GENETICALLY DEFINED
HUMAN GLIOMA MODEL AND A TRANSGENIC MURINE
GLIOMA MODEL
E. Reese,
1
A. Hjelmeland,
1
R. McLendon,
2
E. Holland,
5
D. Bigner,
2

X. Wang,
3
and J. Rich
4
; Departments of
1
Surgery,
2
Pathology,
3
Pharmacology & Cancer Biology, and
4
Medicine, Duke University
Medical Center, Durham, North Carolina;
5
Department of
Neurosurgery, Memorial Sloan Kettering Cancer Center, New York,
New York; USA
Transforming growth factor- (TGF) plays an important role in
gliomas through secretion of angiogenic factors, increased invasion, and
immune escape. We previously demonstrated that TGF potently inhibits
cellular proliferation of astrocytes associated with a G
1
cell cycle arrest
and induction of p15
INK4B
cyclin-dependent kinase inhibitor expression.
This process is dependent on SMAD3, as TGF largely failed to inhibit
the growth of SMAD3/ astrocytes or to induce p15
INK4B
. As most gli-
oma cell lines resist TGF-mediated growth inhibition, we mimicked the
activation of TGF signaling in brain tumors by expressing constitutively
active TGF ligand in a genetically dened glioma model that we have
previously reported. Early passages of this line were growth restricted, but
later passages escaped the antiproliferative effects of TGF and developed
a much greater proliferative potential. Both early and late passage glioma
cultures expressing constitutively active TGF ligand expressed lower levels
of the receptor SMADs (SMAD2 and 3), but the escape from TGF growth
suppression was accompanied only by further SMAD3 expression loss in
later passages. This nding is consistent with reports of decreased SMAD3
expression in human brain tumors. TGF exerts the majority of its effects
through interactions with the microenvironment. Therefore, we modied a
previously developed transgenic glioma model in which an avian retroviral
receptor is expressed in a tissue-specic manner (Nestin tv-a). We inter-
bred the Nestin tv-a mice with Smad3 null mice to determine the impact
of SMAD3 expression on glioma formation. Nestin tv-a SMAD3/ mice
were generated at less than Mendelian ratios but were viable. We generated
tumors through intracranial implantation of retroviruses expressing PDGF
at P
0
. Nearly all mice so treated developed neurological symptoms and
tumors with histopathology similar to human gliomas. The loss of SMAD3
did not impact the latency of tumor development. Despite the limited num-
ber of Smad3/ mice examined, the loss of SMAD3 was associated with
increased tumor grade relative to SMAD3/ and / mice. No SMAD3/
mice lack tumors, whereas other genotypes lacked tumors in a fraction of
mice. Tumor generation continues, and we are characterizing the impact
of targeted disruption of SMAD3 on tumor angiogenesis and invasion. In
conclusion, loss of SMAD3 may contribute to the shift of TGF from a
tumor suppressor to tumor enhancer in gliomas in both human cellular and
transgenic murine glioma models. This work was also supported by NIH
grant NS047409. J.N.R. is a Damon Runyon-Lilly Clinical Investigator and
a Sidney Kimmel Cancer Foundation Scholar.
77. TRAIL IS NONTOXIC TO NORMAL HUMAN
ASTROCYTES
D. Song,
1
J. Song,
1
T. Wilson,
2
V. Yong,
2
and C. Hao
1
; Departments
of
1
Pathology & Laboratory Medicine and Hematology & Oncology,
Emory University, Atlanta, Georgia, USA;
2
University of Calgary,
Clinical Neuroscience, Calgary, Canada
Tumor necrosis factor (TNF) family members such as TNF and FasL
can induce apoptosis in cancer cells; however, their toxicity to normal tis-
sues has limited their usefulness in clinical application. TNF-related apop-
tosis-inducing ligand (TRAIL) is a new member of the TNF family, and its
clinical application currently is under a similar debate. We have reported
that the recombinant soluble TRAIL (amino acids 114-281) can induce
apoptosis in human malignant glioma cells. Here, we report that TRAIL
is nontoxic to normal human astrocytes. Normal human fetal astrocytes
were prepared and cultured in RPMI-1640 medium supplemented with
10% FBS. Cell death was determined by crystal violet assay, and caspase
cleavage was examined on Western blots. Small interfering RNA (siRNA)
was generated to target specic genes. The normal human astrocytes were
treated with FasL and TRAIL, and cell death analysis showed that the astro-
cytes are resistant to TRAIL and FasL-induced cell death. Treatment of the
astrocytes with a pharmacologic inhibitor KN93 to calcium/calmodulin-
dependent protein kinase II (CaMKII) sensitized the astrocytes to FasL-
induced apoptosis through caspase-8-initiated caspase cascade, as evident
by the cleavage of caspase-8, caspase-3 and DNF fragmentation factor 45
(DFF45). Treatment of the astrocytes with KN93 inhibited expression of cel-
lular Fas-associate death domain-like, IL-1b-converting enzyme-inhibitory
protein (c-FLIP) and phosphoprotein enriched in diabetes (PED) in human
astrocytes. Transfection of siRNAs specically targeting c-FLIP and PED
gene sensitized the astrocytes to FasL-induced apoptosis. The results indi-
cate that CaMKII-mediated c-FLIP and PED pathway modulates FasL sig-
naling in human astrocytes. In contrast, neither KN93 nor siRNA targeting
c-FLIP and PED sensitized the human astrocytes to TRAIL-induced apop-
tosis. Flow cytometry analysis revealed cell surface expression of Fas, but
not DR4 and DR5 in the human astrocytes. The study identies the differ-
ent modulation mechanisms between TRAIL and FasL signaling pathways
in normal human astrocytes. Lack of the expression of TRAIL receptors in
human astrocytes contributes to TRAIL resistance.
78. STAGE-SPECIFIC EMBRYONAL ANTIGEN EXPRESSION
IN GLIOBLASTOMA CELLS: POSSIBLE ROLE IN TUMOR
MAINTENANCE
V. Tabar, G. Panagiotakos, and P. Gutin; Department of Neurosurgery,
Memorial Sloan Kettering Cancer Center, New York, New York, USA
Stage-specic embryonal antigens (SSEAs) are a group of carbohydrate
molecules that are characteristically expressed on the surface of embryonic
stem cells during development, or at the undifferentiated stage in vitro.
They are rapidly downregulated at the blastocyst stage and upon ES cell
differentiation. While their role is unclear, this temporal regulation is very
consistent and highly predictive of the differentiation status of ES-derived
progeny. Surprisingly, when we performed histochemical screens on a
group of glioblastoma specimens (n 12), we found consistent expression
of SSEA1, SSEA3, and SSEA4. Gene expression proles in the same group
demonstrate the presence of other genes highly specic to ES cells, such as
Oct4, Nanog and bmi1. To further investigate the parallel expression of
these stem cell-specic factors in ES cells and gliomas, we performed
FACS on several cell groups: undifferentiated human ES cells (NIH WA-01
and WA-09), three different series of human ES-derived neural precursors
(NPC), and cells dissociated from four glioblastomas. Our preliminary data
conrms a very high level of expression of SSEA-4 in the undifferentiated
hES cells (88.17%) while few cells expressed SSEA-1 (0.98%). CD133 was
also signicantly expressed in these cells (59.08%). Upon neural induction
the marker prole is dramatically changed, with downregulation of SSEA-
4 to 3.7%, upregulation of SSEA-1 to 23.18%, with CD133 at 24.06%.
Interestingly, SSEA-4 expression is signicantly upregulated in gliomas
(average of 19.17%), a phenomenon not previously described in somatic
tissues or in other tumors. The levels of SSEA-1 in tumors are somewhat
decreased when compared to NPCs (9.81% vs. 22.67%), and CD133 is
dramatically reduced to 0.2%. Limiting dilution assays were performed on
the fractionated glioma populations. In the one-cell wells a ratio of 1 sphere
per cell was established for the SSEA4 cells vs. 0.5 to 1 for SSEA1 and
0.2 to 1 in the SSEA4- fraction. Subcutaneous injections in NOD/SCID
mice revealed a parallel efciency at tumor formation at dilutions from
500 down to 20 cells/animal/injection. Interestingly, differences among the
positive and negative SSEA4 fractions were not noted upon initial injection
in mice, but rather after the second passage, whereby SSEA4- cells failed to
produce tumors when injected at dilutions of 100 up to 500 cells/injection.
These results were independent of CD133 expression, recently reported as
a cancer stem cell marker, and are suggestive of a complex hierarchy
within glioma tumors, whereby some cell populations (e.g., SSEA4) are
capable of efcient tumor amplication and maintenance parallel to the role
of transit-amplifying progenitors described within the subventricular zone
of the developing or adult brain. Such cells are indistinguishable from stem
cells as currently dened by neurosphere and tumor formation assays.
Additional studies are conducted to further elucidate the expression of
stem cell genes among the populations capable of self-renewal, and the
relationships of SSEA4 expression to that of CD 133.
79. NEURAL STEM/PROGENITOR CELL MIGRATION AND
GLIOMA TARGETING IS STIMULATED THROUGH THE
EGF-PI3K SIGNALING CASCADE
S.E. Kendall,
1
K.S. Aboody,
2
M. Metz,
2
J. Najbauer,
2
E. Garcia,
2
and
C.A. Glackin
1
; Departments of
1
Molecular Medicine and
2
Hematology/
HCT and Neuroscience, Beckman Research Institute, City of Hope
National Medical Center, Duarte, California, USA
Recent advances in neural stem/progenitor cell (NSPC) biology offer
novel strategies to specically target invasive gliomas through chemotac-
tic migration. We have previously demonstrated the inherent tumor-tropic
properties of transduced and untransduced NSPCs to several tumor types.
We are currently utilizing an immortalized human NSPC line, F3.C1
(obtained from Seung U. Kim MD, PhD [UBC]), which targets the inva-
sive U251 invasive experimental glioma in frontal lobe of nude mice. We
hypothesize that EGF produced by the tumor facilitates the NSPC che-
motactic response, showing that a blocking antibody efciently inhibits
chemotaxis toward U251 cells in modied Boyden chamber assays. Fur-
thermore, overexpression of a dominant negative EGFR significantly
reduces the chemotactic response. In contrast, overexpression of a mutant
constitutively active EGFR most commonly associated with high-grade
glioma, EGFRvIII, enhanced NSPC migration irrespective of ligand bind-
ing. The enhanced migration conferred by EGFRvIII on high-grade gliomas
is believed to converge on PI3K signaling. Overexpression of a dominant
negative p85430-615 lacking the p110-binding domain was effective in
blocking migration of F3-EGFRvIII and chemotaxis of F3.C1 cells toward
U251 cells. The activation of PI3K recruits the formation of an active signal-
ing complex at the EGFR cell surface, which includes Shp2. Overexpression
of Shp2 C459S phosphatase dead mutant in F3.C1 cells was effective in
blocking the chemotactic response to U251 and enhanced migration by F3-
EGFRvIII, which suggests that growth factor signaling converges on Shp2
activation to promote cell migration. EGF signals converge on Shp2, and
this leads to subsequent divergence by activating MAPK signaling, cdc42,
and Rac1, while inhibiting RhoA. The inhibitory effects observed through
Shp2 C459S reduce ERK activation and partially reduce the chemotactic
response, which suggests additional routes to enhance cell migration. Con-
stitutively active Shp2 E76A enhances cell migration of F3.C1 cells and
reduces migration with constitutively active RhoA Q63L and thus enhances
actin stress bers. The migratory response is further reduced when cells are
cotransfected with both RhoA Q63L and dominant negative Rac1 N17C.
Collectively, these results demonstrate that the signaling cascade converges
onto PI3K leading to activation of Shp2, thereby providing the necessary
molecular changes to permit chemotaxis toward an EGF gradient.
80. SURVIVAL RATES FOR LOW- GRADE GLIOMA PATIENTS
IN AN INTRA- OPERATIVE MRI
E. Claus, A. Horlacher, L. Hsu, R. Schwartz, D. Dello-Iacono, F. Talos,
F. Jolesz, and P. Black; LEPH, Yale University, New Haven, Connecticut,
USA
No age-and histologic-adjusted assessments of the association between
extent of resection and risk of either recurrence or death exist for neurosur-
gical patients undergoing resection of a low-grade glioma using intraopera-
tive magnetic resonance guidance. The data include 156 patients under-
going surgical resection of a unifocal, supratentorial low-grade glioma in
the magnetic resonance operating suite at Brigham and Womens Hospital
between January 1, 1997, and January 31, 2003. Estimates of disease-free
and overall survival probabilities are calculated by using Kaplan-Meier
methodology. The association between extent of resection and these prob-
abilities is measured by using a Cox proportional hazards model. Observed
death rates are compared to those expected using age- and histologic-
specic survival rates obtained from the Surveillance, Epidemiology, and
End Results Registry (SEER). Patients who underwent subtotal resection
were at 1.4 (95% condence interval [CI], 0.73.1) times the risk of recur-
rence and 4.9 (95% CI, 0.6140.0) times the risk of death relative to patients
undergoing gross total resection. The one-, two-, and ve-year age- and
histologic-adjusted death rates for patients undergoing surgical resection
using intraoperative MRI guidance, 1.9% (95% CI, 0.3%4.2%), 3.6%
(95% CI, 0.4%6.7%), and 17.6% (95% CI, 5.9%29.3%), respectively,
were signicantly lower than those reported using national databases. The
data presented here suggest a possible association between surgical resec-
tion and survival for neurosurgical patients undergoing surgery for a low-
grade glioma under intraoperative MRI guidance. Further study within
the context of a large, prospective, population-based project is needed to
conrm these ndings.
81. TEMPORAL TRENDS IN INCIDENCE OF PRIMARY
GERM CELL TUMORS OF THE BRAIN AND CENTRAL
NERVOUS SYSTEM IN THE UNITED STATES
J. Propp
1
and B. McCarthy
2
;
1
School of Public HealthDivision of
Epidemiology; University of Illinois at Chicago, Chicago, Illinois;
2
Central Brain Tumor Registry of the United States, Chicago, Illinois;
USA
The objective of this study was to describe temporal trends in incidence
of all primary (nonmalignant and malignant) germ cell tumors (GCTs) of
the brain and central nervous system (CNS) in the United States (US) by
using a large population-based series of tumors from the Central Brain
Tumor Registry of the United States (CBTRUS). Brain and other CNS
tumors have increased in the United States over the last two decades, both
overall and for certain histologies. The observed increase is attributable in
part to changes in diagnostic tools and tumor classication and coding. In
the United States, the overall incidence rate for GCT of the brain/CNS is
0.08/100,000 person-years (py) (CBTRUS, 19972001). The rate is high-
est in children 0 to 19 years and young adults 20 to 34 years (0.18 and
0.10/100,000 py, respectively). Rates are higher in males than in females
and in whites than in blacks. CBTRUS compiled data from six state cancer
registries for tumors diagnosed from 1985 to 1999. Multiplicative Pois-
son regression was used to calculate the average annual percent change
(AAPC [95% CI]) in incidence rates over the time period while controlling
for age, sex, race, and microscopic conrmation, and to statistically com-
pare trends over time. Joinpoint regression analysis was utilized to identify
sharp changes in incidence over time. A total of 140 GCTs of the brain/
CNS were diagnosed during the 15-year time period; 76% were in males,
85% in whites, and 90% of the tumors were microscopically conrmed.
Sixty-nine percent were diagnosed in children (019 years), 23% in young
adults (2034 years), and 8% in persons 35 years and older. Incidence of
all primary GCT of the brain/CNS did not signicantly increase over the
time period (AAPC 3.0% [0.9%6.9%]). Incidence rate trends varied
by sex, with females experiencing a signicant increase over the time period
(AAPC 10.7% [2.5%19.6%]) and males experiencing no signicant
change (AAPC 0.8% [3.6%5.2%]). Because GCTs are more common
in the younger compared to the older age groups, we focused our analyses
upon children and young adults. Among children (019 years) there was a
signicant positive time trend (AAPC 5.1% [0.5%9.9%]), and among
young adults (2034 years) there was no signicant change in incidence
over time (AAPC 0.4% (8.6%7.7%)] and no variations in trend by
sex. Data will be presented to best reect signicant temporal trends and/or
sharp changes in incidence that emerged for these tumors. This analysis
adds to the scant literature on trends in incidence of benign and malignant
GCT of the brain/CNS.
82. PRIOR HOSPITALIZATION FOR EPILEPSY AND
SUBSEQUENT RISK OF GLIOMA AND MENINGIOMA
J. Schwartzbaum,
1
F. Jonsson,
2
A. Ahlbom,
2
S. Preston-Martin,
3

B. Malmer,
4
S. Lonn,
2
K. Soderberg,
2
and M. Feychting
2
;
1
Epidemiology
and Biometrics, Ohio State University, Columbus, Ohio, USA;
2
Karolinska Institute, IEM, Epidemiology, Stockholm, Sweden;
3
University of Southern California School of Medicine, Los Angeles,
California, USA;
4
Radiation Sciences, Umea University Hospital, Umea,
Sweden
We conducted a case-control study to evaluate the preclinical associa-
tion between epilepsy and primary adult brain tumors. We rst identied
all 1,501 low-grade glioma (LGG) and 4,587 high-grade gliomas (HGG)
and 4,193 meningioma cases reported to the Swedish Cancer Registry from
1987 to 1999. Next, controls (137,485) were randomly selected from the
continuously updated Swedish Population Registry and matched to cases
diagnosed that year on age and sex. Finally, cases and controls were linked
to the Swedish Hospital Discharge Registry (19691999). We found that
eight or more years before HGG diagnosis (or control reference year) there
was an elevated risk of HGG among people discharged with epilepsy (odds
ratio [OR] 3.01, 95% condence interval [CI], 1.735.22). Two to three
years before HGG diagnosis this risk increased (OR 5.33, 95% CI, 3.58
7.93) and was especially strong among people under age 55 years (OR
13.49, 95% CI, 6.9925.94). During this two- to three-year prediagnostic
period we also found an increased risk of HGG among people discharged
with meningitis (OR 3.02, 95% CI, 1.068.59) or viral encephalitis (OR
12.64, 95% CI, 2.2471.24). Results are similar for glioblastoma mul-
tiforme, LGG, and meningioma. The occurrence of excess epilepsy eight
or more years before HGG diagnosis suggests a relatively long preclinical
phase for these tumors.
83. RECORD OF CENTRAL NERVOUS SYSTEM PRIMARY
TUMORS IN FRANCE: PRELIMINARY RESULTS
L. Bauchet,
1
V. Rigau,
2
H. Mathieu-Daude,
3
D. Figarella-Branger,
4

D. Hugues,
5
M. Fabbro,
6
C. Campello,
7
and F. Segnarbieux
1
;
1
Neurosurgery and
2
Neuropathology, Centre hospitalo-universitaire,
Montpellier;
3
Registre des tumeurs de lHerault, Epidemiology,
Montpellier;
4
Centre hospitalo-universitaire, Neuropathology, Marseille;
5
Groupe de Neurooncologie du Languedoc Roussillon, Epidemiology,
Montpellier;
6
Centre Regional de Lutte Contre le Cancer, Oncology,
Montpellier;
7
Centre Hospitalo-universitaire, Neurology, Nimes; with
the participation of the Association des Neuro-Oncologues dExpression
Franaise, Socit Franaise de Neurochirurgie, and Socit Franaise de
Neuropathology; France
This work aims at prospectively recording all primary tumors (PT) of
the central nervous system (CNS) in France for which histological diagno-
sis is available. The main objectives are to create a national registry and a
network in order to (1) perform epidemiological studies, (2) implement a
new database and use it for setting up both clinical and basic research pro-
tocols, and (3) harmonize the health care of patients affected by CNS-PT.
All French neuropathology and neurosurgery departments have designated
a correspondent to participate to the program. A data le is completed by
the clinician and the neuropathologist for every patient. The le contains
socio-demographic, clinical, radiologic, and anatomopathologic data. The
Tumor Registry from Herault based in Montpellier (south of France), which
is recording data les, has extensive expertise in working with tumor data
and holds the required authorizations for recording nominal data. Over
the rst year, 4094 cases have been recorded from 51 national and private
institutions in France, which includes 53% women and 47% men. Tumor
localizations were supratentorial (80%), infratentorial (14%), and spinal
(7%). Surgical operations were tumor resections (76%) and biopsies (24%).
Histology revealed glioma (48.4%), other neuroepithelial tumors (3.7%),
meningioma (32%), neurinoma (7.8%), lymphoma (3.1%), and others
(5%). Detailed results will be presented during the congress. Our objective
of recording all primary CNS tumor cases nationwide is long and difcult.
During the rst year program, the number of recorded cases has increased.
Preliminary results are encouraging and stimulating for the long-term goal
of creating a National Registry (based on histological data at a rst step)
and a National Network for patients affected by CNS-PT.
84. DESCRIPTIVE EPIDEMIOLOGY OF PRIMARY
OLIGODENDROGLIOMAS IN THE UNITED STATES
J. Propp
1
and B. McCarthy
2
;
1
University of Illinois at Chicago, School
of Public HealthDivision of Epidemiology;
2
Central Brain Tumor
Registry of the United States, Chicago, Illinois; USA
The objective of this study was to estimate the incidence, to describe
temporal trends in incidence, and to estimate survival rates for primary
oligodendrogliomas (OLGs) in the United States. CBTRUS compiled popu-
lation-based data on all primary brain and central nervous system (CNS)
tumors diagnosed between 1997 and 2001 from 15 state cancer registries.
Overall and sex-, race-, and age-specic incidence rates of OLG were esti-
mated. Age-adjusted rates were standardized to the Year 2000 U.S. stan-
dard population. For the analysis of time trends in incidence, CBTRUS
compiled population-based data from 6 state cancer registries for tumors
diagnosed from 1985 to 1999. Multiplicative Poisson regression was used to
calculate the average annual percent change (AAPC [95% CI]) in incidence
rates over the time period while controlling for age, sex, race, and micro-
scopic conrmation, and to statistically compare trends over time. Relative
survival rates for primary OLG for cases diagnosed between 1973 and 2001
in nine Surveillance, Epidemiology, and End Results (SEER) Program areas
were also estimated. Tumors of interest were OLG (ICDO-3 morphology
code 9450/3) and anaplastic OLG (9451/3, 9460/3). Overall incidence rate
of OLG was 0.37/100 000 person-years (py) (CBTRUS, 19972001; N
1585). Median age at diagnosis was 41 years. Rates were higher in males
than in females (0.40 vs. 0.34/100 000 py) and in whites than in blacks
(0.40 vs. 0.14/100 000 py). Rate was lowest in the 0 to19-year age group
(0.08/100 000 py) and highest in the 35- to 44-year age group (0.66/100
000 py). Overall incidence rate of anaplastic OLG was 0.17/100 000 py
(CBTRUS, 19972001; N 738). Median age at diagnosis was 48 years.
Rates were higher in males than in females (0.19 vs. 0.16/100 000 py) and
in whites than in blacks (0.18 vs. 0.09/100 000 py). Rate was lowest in the
0 to 19-year and 85-year and older age groups (0.02/100 000 py) and high-
est in the 55- to 64-year age group (0.32/100 000 py). Incidence of OLG
(N 617) and anaplastic OLG (N 153) increased over the time period
19851999 (AAPC 6.9% [5.1%8.8%]) and 22.3% [17.7%27.2%]),
respectively). One-, ve-, and 10-year survival rates following diagnosis
of OLG were 89%, 70%, and 53%, respectively (SEER; N 1748), and
for anaplastic OLG were 77%, 42%, and 30%, respectively (SEER; N
340). Additional rates by age, sex, and race will be presented. These and
further descriptive epidemiologic studies may facilitate the identication
and elucidation of risk factors for these tumors.
85. RACIAL DISPARITIES IN PATIENT OUTCOME AFTER
CRANIOTOMY FOR TUMORS IN THE UNITED STATES,
19882000
F. Barker, W. Curry, and B. Carter; Department of Neurosurgery,
Massachusetts General Hospital, Boston, Massachusetts, USA
Racially based disparities in American health care are well documented.
We studied disparities in outcomes after 40,101 craniotomies for brain
tumors (primary tumors, metastases, meningiomas, and acoustic neuro-
mas) in the United States, 19882000. The following analytical methods
were used: multivariate proportional-odds ordinal logistic regression cor-
rected for clustering by hospital; random-effects meta-analysis and boot-
strapped heterogeneity estimation. Data source was the Nationwide Inpa-
tient Sample (HCUP, AHRQ, Rockville, Maryland). Analyses adjusted for
age, sex, insurance, income in zip code of residence, geographic region,
admission type and source, medical comorbidity, and hospital volume of
care. In-hospital mortality and adverse discharge disposition were both
more likely in black patients than in others for all tumor types. Odds ratios
for mortality ranged from 1.5 (primary tumors) to 6.3 (acoustic neuro-
mas); combined mortality OR for all tumor types was 1.70 (95% condence
interval [CI], 1.42.1, P < 0.001, no heterogeneity). Odds ratios for adverse
discharge disposition ranged from 1.4 (primary tumors, acoustics) to 1.5
(meningiomas); combined adverse discharge disposition OR for all tumor
types was 1.41 (95% CI, 1.31.6, P 0.001, no heterogeneity). Adjusted
for primary insurance and income in zip code of residence, black patients
were more likely to present as emergency cases: OR, 1.7 (primary tumors)
to 4.6 (acoustic neuromas); combined OR, 2.1, signicant heterogeneity
detected. Medical comorbidity was more severe in black patients: OR 1.3
(metastases) to 2.1 (acoustic neuromas); combined OR, 1.6, signicant
heterogeneity detected. Black patients were signicantly more likely to suf-
fer adverse outcomes after tumor craniotomy in adjusted analyses. Black
patients were also more likely to present as emergency admissions and to
have signicant medical comorbidities than patients of other races, with the
largest disparities observed for benign tumors.
86. PREVALENCE AND PROGNOSTIC SIGNIFICANCE OF
POLYMORPHISMS AT THE GLUTATHIONE
S-TRANSFERASE M1, M3, P1, AND T1 GENE LOCI IN
HUMAN ASTROCYTOMAS
F. Ali-Osman,
1
J.E. Herndon II,
2
L. Stephenson,
1
F. Davis,
4
B.
McCarthy,
4
D. Reardon,
1
A. Friedman,
1
R. McClendon,
5
H. Friedman,
1

and D.D. Bigner
1, 3
; Departments of
1
Surgery,
2
Biostatistics, and
3
Pathology, Duke University Medical Center, Durham, North Carolina;
4
Department of Epidemiology, University of Illinois, Chicago, Illinois;
USA
The glutathione S-transferase (GST) family of genes encodes proteins
that metabolize and inactivate a wide variety of carcinogenic and antican-
cer agents and thus are potential determinants of individual risk to cancer
caused by these agents and of the outcome of chemotherapy. This study
examines the prevalence of polymorphisms at the loci of four GST genes,
namely, GSTM1, GSTM3, GSTP1, and GSTT1, as a function of age, gen-
der, and histological grade in astrocytomas patients and the relationship
of these genetic polymorphisms with patient survival. Genomic DNA from
peripheral blood of 334 patients with grades 1IV astrocytomas was used
to analyze the four GST genes for deletions and/or polymorphisms. Demo-
graphic and GST genotype data were stratied by histological grade and
correlated with patient survival/death. Of the 334 patients, 62% were male
and 38% female. Two thirds had glioblastoma muliforme, 16% anaplastic
astrocytoma, less than 10% low-grade astrocytoma. Patients of age 50
years had a 2-fold higher rate of glioblastoma compared to those under
50 years, while lower grade tumors were more prevalent in the under 50-
year group. GSTM1 null and positive genotypes were present in equal pro-
portions. Among the GSTM1ve patients, the GSTM1A allele was 2-fold
more frequent than GSTM1B, with GSTM1A/B heterozygosity in only 5%.
The GSTM3 gene was deleted in 2% of the patients, and the GSTM3A
allele was 4-fold more frequent than the M3B allele. The GSTP1A allele
(Ile104/Val113) was present at 66%, GSTP1B (Ile104/Ala114) at 29%, and
GSTP1C (Val104/Val113) at 9%. Despite its low frequency, the GSTP1C
allele was 2-fold more prevalent in glioblastoma patients than in others. No
relationship was observed between GSTM1 and GSTM3 polymorphisms
and survival. These analyses of the GSTM1A/B alleles and the GSTM3
genes are the rst to be reported for malignant gliomas. In contrast, the
GSTP1C allele was associated with early death. We conclude that signi-
cant differences exist in the prevalence of GST alleles and genotypes in
different histological grades of astrocytomas and that the GSTP1C and
GSTT1 null genotypes are associated with decreased survival and early
death in this tumor type. The ndings provide novel insights into the poten-
tial signicance of these polymorphisms and into the possible mechanistic
role of these genes in astrocytomas and are an important contribution to
efforts at dening the role of genetic polymorphisms in brain tumor etiol-
ogy and prognosis. This research was supported by grants RO1 CA91438,
RO1 CA79644, and P50-CA108786 from the NIH (USA).
87. IS ANDROPAUSE PROTECTIVE IN THE DEVELOPMENT
OF MENINGIOMAS IN MEN?
J. Grutsch
1
, C. Lis
1
, J. Propp
2
, B. McCarthy
2
, and F. Davis
2
;
1
Cancer
Treatment Centers of America, Zion, Illinois;
2
University of Illinois at
Chicago, Epidemiology and Biostatistics, Chicago, Illinois, USA
Clinical, epidemiological, and hormonal receptor data imply a link
between meningiomas and ovarian hormones. The fact that a third of
meningiomas occur in men and active androgen receptors are found in
nearly 70% of tumors arising in males suggests that there may be addi-
tional hormonal inuences. We indirectly investigated the protective effect
of andropause on the age-specic incidence of meningiomas arising within
the cerebral hemispheres and spine by evaluating the relationship between
age at diagnosis and meningioma. Population-based incidence data from
the Central Brain Tumor Registry of the United States (CBTRUS) was
obtained, and linear regression methods were applied. The rate of increase
in incidence rates accelerated in males after age fty-ve. Age-specic linear
regressions found that the slope of the line under age fty-ve was 2.5 and
for those over age fty-ve was 3.8. Considering tumor location, there was
no exponential increase in spinal meningioma rates until age 50 where, up
to age 75, the slope was 5.2. The risk of male meningioma also accelerates
after age fty in the cerebral hemispheres. These male increases, rather
than a female decrease, account for the declining female/male ratios in
patients over 55 years of age. These results suggest the existence of multiple
genetic/etiologic pathways leading to the development of meningiomas. In
addition, the etiology of meningiomas may be contingent upon gender and
anatomical location.
88. THE ROLE OF ENVIRONMENTAL AND HORMONAL
FACTORS IN THE ETIOLOGY OF RADIATION-ASSOCIATED
AND SPORADIC MENINGIOMAS
S. Sadetzki, P. Flint-Richter, and B. Oberman; Cancer and Radiation
Epidemiology, Gertner Institute, Sheba Medical Center, Ramat-Gan,
Israel
Current knowledge about the etiology of meningiomas is sparse, with
ionizing radiation being the only well-established risk factor. The aim of
this study was to comprehensively assess the contribution of environmen-
tal and hormonal risk factors in radiation-associated meningiomas (RAM)
and sporadic meningiomas. Between 1948 and 1960 about 20,000 children
in Israel were treated with ionizing radiation to the scalp for tinea capitis
(TC). An additional unknown number of children were treated abroad.
This research was designed as a nested 4-group case-control study balanced
for irradiation that enabled the estimation of the main effect of each sus-
pected risk factor as well as its interaction with radiation. The total study
population included 526 subjects: 161 RAM cases that were irradiated for
TC in childhood and subsequently developed meningioma, 85 sporadic
cases, 145 irradiated controls, and 135 nonirradiated controls. The latter
3 groups were individually matched to the RAM cases by gender, year of
birth, and continent of origin. Data on smoking, alcohol consumption, his-
tory of head trauma and asthma, parity, age at menarche and menopause,
and use of exogenous hormones was collected by using a personal interview.
In a multivariate analysis of risk factors in men, smoking was associated
with a signicant increased risk for meningioma (OR 2.50; 95% CI,
1.195.27), while alcohol consumption decreased the risk (OR 0.12;
95% CI, 0.020.87). For both of these factors a dose-response association
was found. In women, a signicant interaction between active smoking and
radiation was observed. In the subgroup of nonirradiated women, smoking
was associated with a decreased risk for meningioma (OR 0.34; 95%
CI, 0.130.88. The risk decreased with increasing smoking pack-years (P
for trend 0.003). This protective effect was not seen among irradiated
women. In the analysis of hormonal factors, we observed a protective effect
for variables that reect a reduced exposure to endogenous and exogenous
feminine hormones. This study design enabled the assessment of risk factors
(other than radiation) for meningiomas and the evaluation of interactions
between radiation and other determinants in the development of this tumor.
The dose-response effect observed for both smoking and alcohol in men
supports the causal interpretation of these results. The protective effect of
smoking in nonirradiated women may be explained by the antiestrogenic
effect of smoking. The negative association found between exposure to
feminine hormones and meningiomas is biologically plausible, considering
the higher incidence of this tumor in women and the existence of steroidal
hormone receptors in the tumor tissue. As more mechanistic information
on susceptibility and etiology of the disease become available, risk assess-
ment and possible primary and secondary prevention could be considerably
improved.
89. POLYMORPHISMS ASSOCIATED WITH ASTHMA ARE
INVERSELY RELATED TO GLIOBLASTOMA MULTIFORME
RISK
J. Schwartzbaum,
1
A. Ahlbom,
2
B. Malmer,
3
S. Lonn,
2
A. Brookes,
4

W. Debinski,
6
R. Henriksson,
3
and M. Feychting
2
;
1
Epidemiology and
Biometrics, Ohio State University, Columbus, Ohio, USA;
2
Epidemiology,
Karolinska Institute, IEM, Stockholm, Sweden;
3
Radiation Sciences,
Umea University Hospital, Umea, Sweden;
4
Genetics, University
of Leicester, Leicester, England;
5
Statistics, Ohio State University,
Columbus, Ohio, USA;
6
Brain Tumor Center of Excellence, Wake Forest
University, Winston-Salem, North Carolina, USA
A reduced risk of primary adult brain tumors is observed among people
reporting asthma, hayfever, and other allergic conditions; however, nd-
ings may be attributable to prediagnostic effects of tumors or recall bias.
To determine whether asthma and allergic condition polymorphisms are
inversely related to glioblastoma multiforme (GBM) risk, we conducted a
population-based case-control study of 111 GBM patients and 422 controls.
We identied ve single nucleotide polymorphisms (SNPS) on three genes
previously associated with asthma (interleukin [IL] 4RA, IL13, ADAM33)
and one gene associated with inammation (COX2). Conrming previ-
ous literature, we found that self-reported asthma, hayfever, and eczema
reduce GBM risk (e.g., asthma and hayfever at time of interview, odds
ratio [OR] 0.38, 95% condence interval (CI), 0.180.83). In addition,
IL4RA ser478pro TC, CC and IL4RA gln551arg AG, AA are associated
with increased GBM risk (OR 1.64; 95% CI, 1.052.55; and 1.61; 95%
CI, 1.052.47) while IL13 -1112 CT, TT is associated with decreased GBM
risk (0.56, 95% CI, 0.330.96). Each of these polymorphism-GBM associ-
ations is in the opposite direction of a corresponding polymorphism-asthma
association, thereby conrming previous ndings that asthmatics and peo-
ple with allergic conditions have lower GBM risk than do people without
these conditions. Because we used germline polymorphisms as biomarkers
for asthma and allergic conditions, our results cannot be attributed to recall
bias or effects of GBM on the immune system. Our ndings have implica-
tions for asthma or allergy treatments that inhibit IL13 production.
90. FEASIBILITY OF WEB-BASED VERSUS TELEPHONE
INTERVIEWS AS A DATA COLLECTION METHOD IN
EPIDEMIOLOGY STUDIES OF BRAIN TUMOR PATIENTS
G. Rauscher,
1
K. Rankin,
1
S. Erdal,
2
B. McCarthy,
1
and F. Davis
1
;
1
Epidemiology and Biostatistics, and
2
Environmental and Occupational
Health Sciences, University of Illinois at Chicago, Chicago, USA
As part of a pilot study conducted for a large case-control study of neu-
rocarcinogens, gene polymorphisms, and adult brain cancer, we have devel-
oped a Web-based data collection instrument intended to allow patients to
participate at their own pace in a comfortable environment, while minimiz-
ing study data collection effort. Among 223 respondents who completed the
Web-based survey and 67 respondents who completed a telephone survey,
we examined characteristics associated with the choice of survey mode. We
also examined the reliability of responses for 140 respondents who com-
pleted both the Web-based survey and a short Web-based resurvey, as well
as the reliability of responses for 47 respondents who completed both a
phone survey and phone resurvey. Compared to telephone survey partici-
pants, Web-based survey participants were less likely to be divorced or wid-
owed (7% vs. 20%, respectively), more likely to have family incomes over
100,000 (40% vs. 23%), and less likely to have never searched the Internet
(4% vs. 24%). Individuals completing the Web-based survey, however, were
also slightly more likely to perceive the interview as difcult to complete
(9% vs. 5%). The reliability (Kappa or Spearman rank correlations) for
responses to 5 questions on history of smoking and oral contraceptive use
ranged from 0.84 to 0.97 for Web responses and 0.56 to 0.92 for phone
responses. Responses to questions intended for use in constructing vari-
ables for exposures to neurocarcinogens were less reliable. These included
questions on living arrangements (heating sources, drinking water sources,
dwelling type, living on a farm, daycare as a child), methods of food prepa-
ration, intake of fresh fruits and vegetables, and use of indoor pesticides.
Reliability for the 23 exposure variables examined ranged from 0.17 to
0.78 (median, 0.54) for Web responses and from -0.02 to 1.0 (median,
0.51) for phone responses. Phone respondents answered a median of 8 of
35 questions with concordance at both surveys, whereas Web respondents
answered a median of 23 of 35 questions with concordance at both surveys.
Results will be presented by case and control status and controlled by age.
Preliminary results suggest that a Web-based survey format is feasible in
some research settings and that it has some advantages over traditional
formats. However, to obtain information on all demographic subgroups
and to minimize biases in studies, more than one mode of data collection
may be optimal.
91. AGE-RELATED PATTERNS IN CNS TUMOR INCIDENCE
WITH A FOCUS ON THE ADOLESCENT AND YOUNG
ADULT POPULATION
D. Walker,
1
C. Stiller,
2
L. Ries,
3
A. Bleyer,
4
and A. Bendel
5
;
1
Childrens
Brain Tumour Research Centre, University of Nottingham, Nottingham,
UK;
2
Childhood Cancer Research Group, Oxford, UK;
3
National Cancer
Institute, Bethesda, Maryland, USA;
4
The University of Texas M.D.
Anderson Cancer Center, Houston, Texas, USA;
5
Childrens Hospital &
Clinics, Minneapolis, Minnesota, USA
Brain tumor incidence data were analyzed from two large population-
based databases, the Surveillance, Epidemiology, and End Results Pro-
gram (SEER) and the Central Brain Tumor Registry of the United States
(CBTRUS), to determine the incidence of brain tumors according to histo-
logical subtype and age at diagnosis. The focus of the study was to analyze
incidence of CNS tumors in the adolescent and young adult (AYA) popula-
tion and to look for any patterns of incidence that might explain mechanisms
of tumorigenesis. Incidence of CNS tumors during 1975 to 1998 in SEER
registries was determined for each 5-year age group from 0 to 44 years.
SEER groups CNS tumors into broad histological categories: astrocytomas
(grade IIV), other gliomas (oligoastrocytoma and oligodendroglioma),
PNET, ependymoma and germ cell tumors. Additional incidence data were
obtained from the published 19951999 statistical report of the CBTRUS,
to supplement the SEER data, and to provide age-related incidence data for
specic histological subtypes, including meningioma, craniopharyngioma,
mixed glioma, and each grade of astrocytoma and oligodendroglioma. Four
patterns of tumor incidence were recognized from the SEER and CBTRUS
databases: (1) peak incidence in the 15-to 19-year age group (germ cell
tumors), (2) falling incidence with aging (grade I astrocytoma and PNET),
(3) rising incidence with aging (grade IIIV astrocytoma, oligodendro-
glioma, oligoastrocytoma and meningioma), and (4) stable incidence with
aging (craniopharyngioma and ependymoma). The pattern of age-related
incidence of CNS tumors suggests that their development is driven, in part,
by factors linked to the completion of the brains growth and development,
by hormones key to sexual maturation, and by factors that inuence adult
aging. The peak incidence of germ cell tumors in the AYA years justies
them being taken as the model tumor for AYA neuro-oncology. Their rar-
ity justies a worldwide clinical trial strategy building upon evidence of
rising survival rates (germinoma 90%, nongerminomatous germ cell
tumor 60% 10 year). Quality of survival is widely recognized as a prior-
ity, although it has not yet been selected as a primary outcome measure. We
propose a worldwide, combined age approach to the investigation of CNS
germ cell tumors aimed at optimizing quality of survival.
92. EPIDEMIOLOGICAL AND CYTOGENETICAL
INVESTIGATIONS IN THE GROUP OF THE CHERNOBYL
CLEAN-UP WORKERS WITH THE DISEASES OF NERVOUS
SYSTEM
E. Diomina, N. Ryabchenko, E. Smekhova, and M. Diomina;
Department of Radiobiology, R.E. Kavetsky Institute of Experimental
Pathology, Oncology and Radiobiology, Kyiv, Ukraine
The increase of the case rate of malignant pathologies in Ukraine is
signicantly connected with the rise of the cancerogenic loading on the
population. Special attention to the cancerogenic effects of radiation was
given after the accident in Chernobyl. The purpose of the present investiga-
tion was to study the frequency of the diseases of nervous system (cancer
and noncancer pathologies), peculiarities of cytogenetical effects in blood
lymphocytes of clean-up workers of the Chernobyl catastrophe, and their
dependence on the dose of radiation exposure. Epidemiological studies
(more than 20,000 clean-up workers with documented doses of exposure)
and metaphase analyses of radiation-induced chromosome aberrations in
peripheral blood lymphocytes (cytogenetical investigation of 2000 clean-
up workers with documented doses of irradiation) were conducted. It
was established that diseases of nervous system took the rst place in the
structure of the development of diseases of the clean-up workers of the
Chernobyl catastrophe during all years of our investigation (19902002).
Statistically signicant tendency of the increase of the diseases of ner-
vous system (unstochastic effects) with the dose of irradiation (1 85 cGy)
was observed irrespectively of the age of clean-up workers. As for neuro-
oncological pathologies (stochastic effects), their highest frequency was reg-
istered in the group of the clean-up workers irradiated in a range of low doses
(15 cGy). Dose dependence of the frequency of radiation markers (dicen-
tric chromosomes) in the lymphocytes of peripheral blood was observed
only in a group of clean-up workers with neuro-oncological pathologies,
when the lack of anticancer protection of the organism was caused by the
radiation injury in low doses. Cytogenetical criteria for the formation of the
groups with the increased neuro-oncological risk and the improvement of
the monitoring of the health status of clean-up workers were determined.
Low doses of ionizing radiation are statistically signicant factors of cancer
risk, including development of neuro-oncological pathologies.
93. THE RISK FOR MALIGNANT PRIMARY ADULT- ONSET
GLIOMA IN A LARGE, MULTI-ETHNIC, MANAGED CARE
COHORT: CIGARETTE SMOKING AND OTHER LIFESTYLE
BEHAVIORS
J.T. Erd,
1
G.D. Friedman,
2
S. Sidney,
3
A. Klatsky,
3
L.A. Haurel,
3

N.V. Udaltsova,
3
S. Van Den Eeden,
3
and L.M. Nelson
2
;
1
John A.
Burns School of Medicine, Ofce of the Dean, Honolulu, Hawaii;
2
Stanford School of Medicine, Health Research and Policy, Division of
Epidemiology, Palo Alto, California;
3
Kaiser Permanente Medical Care
Program, Division of Research, Oakland, California; USA
The purpose of this study was to determine the risk for malignant pri-
mary adult-onset glioma (MPAG) associated with cigarette smoking and
other lifestyle behaviors in a large, multi-ethnic, managed-care cohort. The
study population included a cohort of 133,811 subscribers to the Kaiser
Permanente Medical Care Program of Northern California (KPMCP-NC)
who had received a multiphasic health checkup and questionnaire (MHC)
between 1977 and 1985, were at least 25 years old at their start of follow-
up, and had no prior history of benign or malignant brain tumors. In this
cohort, patients were followed for up to 21 years for the development of
MPAG. Risk for MPAG among women increased with increasing packs
of cigarettes smoked per day (P for trend 0.04), adjusting for cigar and
pipe smoking, patient age, sex, race, education, alcohol use, and coffee
consumption. A similar pattern was not observed for men. Individuals who
smoked marijuana at least once a month, adjusting for cigarette smoking
(packs smoked per day) and for the factors noted above, had a 2.8 (CI,
1.36.2)-fold increased risk for MPAG. Relative risk for MPAG increased
with increasing consumption of coffee (P for trend 0.05). Cigarette smok-
ing was associated with an increased risk for MPAG among women but
not among men. Individuals who smoked marijuana at least once a month
had an increased risk for MPAG, although no dose-response relation was
observed. Drinkers of 7 cups of coffee per day had a 70% increased risk
for MPAG and smaller risk elevation for lower consumption. Alcohol use
was not associated with an increased risk for MPAG.
94. MATERNAL DIET DURING PREGNANCY AND ITS
ASSOCIATION WITH MEDULLOBLASTOMA
G.R. Bunin,
1
L.H. Kushi,
2
P.R. Gallagher,
3
L.B. Rorke-Adams,
4

M.L. McBride,
5
and A. Cnaan
3
;
1
Oncology, Childrens Hospital of
Philadelphia, Philadelphia, USA;
2
Research, Kaiser Permanent, Oakland,
California, USA;
3
Biostatistics and Epidemiology and
4
Pathology,
Childrens Hospital of Philadelphia, Philadelphia, Pennsylvania, USA;
5
Cancer Control Research Unit, British Columbia Cancer Agency,
Vancouver, Canada
Fruit, vegetables, vitamin C, and folate during pregnancy have been
suggested as protective factors for medulloblastoma, a common brain tumor
in children. The authors sought to replicate these ndings and investigate
other aspects of diet. Mothers of 315 cases under age six at diagnosis and
of 315 controls were interviewed about their pregnancy diet. The authors
observed modest, inverse associations for fruits/juices (odds ratio [OR] for
highest compared to lowest category 0.6, 95% condence interval [CI],
0.31.1) and vitamin C (OR 0.6; 95% CI, 0.41.1). In contrast to the pre-
vious study, folate and vegetables showed no association. As hypothesized,
cured meats were not associated with medulloblastoma, unlike previous
ndings for other childhood brain tumors. An inverse association with
non-fresh peaches and similar fruits (OR 0.5; 95% CI, 0.30.8) and a
positive association with nonchocolate candy (OR 1.7, 95% CI, 1.03.0)
replicated previous ndings. Vegetable fat (OR 1.7; 95% CI, 1.02.9)
and French fries (OR 2.4, 95% CI, 1.24.9) were associated with medul-
loblastoma. The lack of association with cured meats suggests that medullo-
blastoma differs in etiology from astrocytoma (for which associations with
cured meats have been observed in several studies). The results for vitamin
C, fruit/juices, non-fresh peaches, and candy replicate previous ndings for
medulloblastoma and suggest that these aspects of diet are worthy of more
detailed investigation.
96. ORGANOTYPIC SPHEROIDS OF GLIOBLASTOMA: A
NOVEL HIGH-THROUGHPUT DRUG SCREENING SYSTEM
P. De Witt Hamer,
1
A. Van Tilborg,
1
C.J.F. Van Noorden,
2
A.H.
Zwinderman,
3
D. Troost,
4
and S. Leenstra
1
;
1
Neurosurgery,
2
Cell
Biology & Histology,
3
Clinical Epidemiology & Biostatistics, and
4
Neuropathology, Academic Medical Centre, Amsterdam, The
Netherlands
The plethora of newly developed potential therapeutics for glioblas-
toma requires a test model that adequately quanties response and is bio-
logically valid. Historically, monolayer cultures and commercially available
cell lines are used for this purpose. However, there is marked discrepancy
in responses of these widely used test systems and human tumor responses,
which illustrates the biological invalidity of these cell-suspension test
models. This is partially explained by clonal selection and loss of inter-
cellular cross-talk. To circumvent these problems, surgical specimens are
cultured in medium as explants on the short term and grown as organo-
typic spheroids as previously described by Bjerkvig et al (J. Neurosurg. 72,
463, 1990). Limitations for use in drug experiments were based on (1) lack
of quantitation of response using only qualitative morphological assess-
ment, (2) heterogeneity of spheroids with undetermined implications for
design of experiments, and (3) only hypothesized superior biological valid-
ity as compared with widely used and easily quantitated cell-suspension
models. First, therefore, a semiautomated method has been developed that
facilitates quantication of viability, proliferation, and apoptosis, and this
appears to be a valid quantitative approach to determine response. Images
of enzyme-histochemically and immunohistochemically stained cryostat
sections of spheroids are digitally captured and analyzed using automated
image cytometry. Second, the natural variance of measurements dictates
the number of spheroids to be used in experiments. This is shown to be
within ranges that allow for high-throughput screening. Third, to demon-
strate the biological validity of the spheroid test model, DNA microarrays
experiments are performed to compare the genetic prole of the original
tumor with both monolayer culture and organotypic spheroids from the
same material. It is hypothesized that the organotypic spheroids show good
correlation with the original tumor, in contrast to the monolayer culture. In
conclusion, an organotypic spheroid test system for in vitro drug screening
in malignant glioma has been developed that rapidly quanties response,
allows for high-throughput screening, and appears to be superior to cell-
suspension models in genetic prole as compared with the original tumor.
97. MOLECULAR PROFILING AND CHARACTERIZATION
OF SYNTHETIC ASTROCYTES, DIFFERENTIATED FROM
EMBRYONIC STEM CELLS IN VITRO
D. Kamnasaran
1
and A. Guha
1,2
;
1
Neurosurgery Division, Toronto
Western Hospital;
2
Neurosurgery Division, Labatts Brain Tumor Centre,
HSC, Toronto; Canada
The pluripotency of embryonic stem (ES) cells is determined by the
ability to differentiate into derivatives of the three germ layers. We have
determined an optimal protocol for the in vitro synthesis of astrocytes from
murine ES cells harboring wild-type (wt) and p53 () genetic backgrounds
and undertaken expression and tumorigenicity studies. Astrocytic differen-
tiation of the ES cells, with GFAP positive and negative OLIG2, NESTIN
plus OCT2 expression, was shown in more than 90% of cells. RT-PCR
for these and other neural-glial lineage markers further supported that
these were synthetic derived astrocytes from the ES cells. The majority of
the synthetic astrocytes from wt-ES cells displayed morphology similar to
Type-1 astrocytes. cDNA microarray analyses on Affymetrix arrays were
performed. After normalizing and scaling of the data, the transcriptomes
of synthetic astrocytes with wt or p53 () genetic backgrounds were found
to be very similar (r 0.89). However, several genes were differentially
expressed (P 0.001), most likely representing p53 response genes. For
some of these differentially expressed genes, verication was obtained by
real-time PCR. The transcriptomes of the synthetic astrocytes were com-
pared to the published transcriptomes of in vivo astrocyte cultures estab-
lished from various murine embryonic, postnatal, and adult brain sections
(PNAS 101, 8384). We calculated the extent of similarity (Pearsons corre-
lation coefcient (r), P 0.001) with possible astrocyte specic genes (~325
transcripts) identied by having an expression pattern similar to GFAP,
using a recursive-supervised machine (R-SVM) class prediction analysis.
Wt- synthetic astrocytes were found most similar to astrocytes from the
adult corpus callosum (r 0.281), while synthetic p53 () astrocytes were
most similar to astrocytes from the cortex (P2) (r 0.212). By compari-
son, using 156 astrocyte-specic markers identied by R-SVM analysis,
both wt- and p53 () synthetic astrocytes were found to be most similar
to astrocytes from the cortex (P2) (r 0.287, r 0.315). Finally, both
wt- and p53 () synthetic astrocytes were tested for in vivo intracranial
transformation in Nod-Scid mice. Intracranial injections into these mice
with wt- or p53 () ES cells resulted in teratomas within 3 to 5 weeks. No
tumors developed with wt- or p53 () synthetic astrocytes. Study is ongoing
with synthetic derived astrocytes from ES cells, of varying glioma relevant
genetic backgrounds, which we believe provide a relatively quick bioassay
to understand the roles of these genetic alterations in glial differentiation
and transformation.
98. REGIONAL BRAIN SPHEROID CULTURE: AN IN
VITRO 3D MODEL TO STUDY SPECIFICITY OF TUMOUR
INVASION
T.L. Parker,
1
K. Searle,
1
L. Storer,
2
and D. Walker
2
;
1
Institute of
Neuroscience, School of Biomedical Sciences, University of Nottingham
Medical School, Queens Medical Centre, Nottingham;
2
Division of
Child Health, University of Nottingham, Nottingham; UK
Dimensionality plays a major role in cellular behavior, and in vitro
tumor invasion studies have been hampered by the lack of suitable 3D
models of tumor/host brain interaction. Allowing enzyme dissociated cells
to reform into 3D spheroids has provided the means of developing such a
model. Spheroid cultures of enzymatically dissociated fetal rat cerebellum,
brainstem, and cerebral cortex (E18) were morphologically and immuno-
histochemically characterized at 3, 5, and 10 days in vitro. Comparisons
between 3D spheroids, 2D monolayer, and undissociated brain tissue dem-
onstrate that spheroid cultures begin to differentiate and express neurola-
ment proteins and glial brillary associated proteins (GFAPs) in addition to
forming synaptic contacts and extracellular collagen. Neural cells appear
to differentiate at a higher rate than glial. The developmental prole of
neurons and glia in the 3D regional brain spheroids mimics the in vivo
cytoarchitecture to a greater extent than 2D brain cell cultures. The level
of GFAP immunoreactivity (IR) is signicantly greater than neurolament
(NF) immunoreactivity (IR) in all monolayer cultures (50%), compared
to 8.3% NF in vitro. Levels do not signicantly change over time in vitro.
NF IR reaches 97% and GFAP 65%. Adult rat brain tissue has a signi-
cantly greater IR to NF (88%) than GFAP (51%). Therefore, the ratio of
NF:GFAP IR in regional brain spheroids approaches that found in adult
rat tissue. The 3D spheroid microenvironment results in cellular selection
that more closely reects adult tissue cellular composition. By using a 3D
spheroid co-culture model of host brain/Daoy cells (medulloblastoma cell
line), it was found that the Daoy cells preferentially invaded cerebellum and
brainstem, and not the cerebral cortex, possibly reecting differing regional
cellular microenvironments. Therefore, this 3D model appears to mimic in
situ medulloblastoma behavior. The viability and reproducibility of these
regional brain spheroid cultures make them a useful tool for further inves-
tigations into developmental biology, neurotoxicity, and antitumor therapy
in different brain regions.
99. HEMATOPOIETIC STEM CELLS HOME TO MALIGNANT
GLIOMAS
G. Tabatabai,
1
O. Baehr,
1
R. Moehle,
2
I. Eyuepoglu,
3
A. Boehmler,
2

J. Wischhusen,
1
J. Rieger,
1
I. Bluemcke,
4
M. Weller,
1
and W. Wick
1
;
1
Laboratory of Molecular Neuro-Oncology, Department of General
Neurology and Hertie Institute for Clinical Brain Research, Tuebingen;
2
Department of Internal Medicine II, University of Tuebingen,
Tuebingen;
3
Departments of Neurosurgery and
4
Neuropathology,
University of Erlangen, Erlangen; Germany
Stem and progenitor cells (PCs) of various lineages have become attrac-
tive vehicles to improve therapeutic gene delivery to cancers, notably glio-
blastoma. Here we report that adult human and murine hematopoietic PCs
display a tropism for intracerebral gliomas but not for normal brain tissue
in mice. Organotypic hippocampal slice culture and spheroid confrontation
assays conrm a directed PC migration toward glioma cells ex vivo and
in vitro. RNA interference-mediated disruption of transforming growth
factor beta (TGF-b) synthesis by the glioma cells strongly inhibits PC cell
migration. We delineate a CXC chemokine ligand (CXCL) 12dependent
pathway of TGF-b-induced PC migration that is facilitated by MMP-9-
mediated cleavage of stem cell factor (SCF). Moreover, neutralizing anti-
bodies to CXCL12 strongly reduce PC homing to experimental gliomas in
vivo. Thus, we dene here the molecular mechanism underlying the glioma
tropism of the probably most easily accessible PC population suitable for
cancer therapy, that is, adult hematopoietic PC. This work was supported
by the Landesstiftung Baden-Wrttemberg, State of Baden-Wrttemberg,
Germany (P-LS-AS/HSPA7-12).
100. ACTIVATION OF PROTEOLYTIC PATHWAYS BY
POLYPHENOLIC PHYTOCHEMICALS FOR APOPTOSIS IN
HUMAN GLIOBLASTOMA T98G CELLS
S. Ray,
1
A. Das,
1
S. Karmakar,
1
S. Patel,
2
and N. Banik
1
; Departments of
1
Neurology and
2
Neurosurgery, Medical University of South Carolina,
Charleston, South Carolina, USA
Glioblastoma, the deadliest and most prevalent human brain tumor,
still remains refractory to almost all conventional chemotherapeutic agents.
Therefore, alternative and innovative therapeutic strategies need to be
developed for treating this deadly disease in humans. Extensive research
in the last few years has revealed that regular consumption of certain fruits
and vegetables can reduce the risk of acquiring specic cancers. Flavonoids
such as apigenin (APG), (-)-epigallocatechin (EGC), (-)-epigallocatechin-
3-gallate (EGCG), and genistein (GST) are polyphenolic phytochemicals,
which ubiquitously occur in fruits and vegetables. Flavonoids have been
shown to suppress proliferation of various cancer cells, inhibit growth
factor signaling pathways, suppress expression of antiapoptotic proteins,
and also induce apoptosis, indicating that they may have untapped thera-
peutic value. Very recently, these phytochemicals have also been found to
reverse both radioresistance and chemoresistance in patients undergoing
glioblastoma treatment. In the current investigation, we have examined the
molecular events relating to mitochondria-mediated apoptosis in human
glioblastoma T98G cells following exposure to APG, EGC, EGCG, and
GST. We used trypan blue dye exclusion test for assessing cell viability,
Wright staining for determining distinct morphological characteristics of
apoptosis, modied version of the telomerase repeat amplication protocol
(TRAP) assay for detecting telomerase activity, fura-2 assay for quantifying
intracellular free [Ca
2
], Western blottings for examination of expression
of specic proteins, and also a colorimetric assay for estimating caspase-3
activity in T98G cells following exposure to the avonoids. Trypan blue dye
exclusion test showed that the number of viable T98G cells was decreased
in a dose-dependent manner when T98G cells were exposed to these phyto-
chemicals. Wright staining indicated predominantly apoptotic morphology
in T98G cells exposed to 100 mM APG, 50 mM EGC, 50 mM EGCG, or
100 mM GST, for 24 h. We applied a modied version of the TRAP assay
to phytochemical treated T98G cells to determine any downregulation in
the activity of telomerase, an enzyme responsible for lending an unlimited
capability of proliferation to the cancers including glioblastoma. Apoptosis
in T98G cells following exposure to these phytochemicals was associated
with an increase in intracellular free [Ca
2
], as determined by fura-2 assay.
Western blot analyses showed an increase in Bax:Bcl-2 ratio, as well as
overexpression of calpain and caspase-3 that were also activated to cleave
270 kD a-spectrin at specic sites for generation of 145 kD spectrin break-
down product (SBDP) and 120 kD SBDP, respectively. Further, colorimetric
assay using a synthetic substrate specic for caspase-3 conrmed activation
of caspase-3 in apoptotic T98G cells following exposure to 100 mM APG,
50 mM EGC, 50 mM EGCG, or 100 mM GST for 24 h. Taken together,
these results

strongly suggest that selective polyphenolic phytochemicals
can be used for activation of proteolytic activities of calpain and caspase-3
for apoptosis in human glioblastoma T98G cells. This investigation was
supported in part by the R01 grants from the NCI and NINDS of the NIH
and also a grant from the State of South Carolina.
101. COMBINATION OF HISTONE DEACETYLASE
INHIBITORS AND RADIATION OFFERS A PROMISING
THERAPY FOR NEUROBLASTOMA
K. Hovinga,
1
M. Entin-Meer,
1
X. Yang,
1
N. Jelluma,
1
J. Chen,
1
W.
Weiss,
2
L. Chesler,
2
A. Nudelman,
3
A. Rephaeli,
3
and D. Haas-Kogan
1
;
1
Department of Radiation Oncology and Comprehensive Cancer Center
and
2
Department of Neurology, Pediatrics, and Neurological Surgery,
University of California San Francisco, San Francisco, California, USA;
3
Pharmacology and Experimental Oncology, Felstein Medical Research
Center, Tel Aviv, Israel
Neuroblastoma, a malignancy arising from neural crest cells, exhibits
resistance to current therapies. HDAC inhibitors have emerged as exciting
new cancer therapies and have demonstrated antineoplastic effects against
neuroblastoma. Pivaloyloxymethyl butyrate (AN-9, Pivanex) was derived
from the HDACI butyric acid (BA) and shows more efcient delivery and
higher intracellular concentrations in comparison with BA. We sought to
test the combination of the AN-9 and radiation in the treatment of neu-
roblastoma in vitro. The neuroblastoma cell line SHEP21, constructed to
contain inducible MYCN, was treated with different doses of radiation
and AN-9. Effects on cell viability and apoptosis of AN-9, radiation. and
combinations thereof were tested in high and low Mycn-expressing cells
by using MTS assays and FACS analyses. The effect of AN-9 on the Mycn
expression was examined by Western blot. We also addressed the inuence
of the order of administration of AN-9 and radiation on antineoplastic
activity. Consistent with published literature, AN-9 inhibited Mycn expres-
sion. Combinations of AN-9 and radiation were more effective than either
treatment alone. The combined effect was additive in MTS cell viability
assays. FACS analyses demonstrated that high-Mycn-expressing cells were
markedly more susceptible to apoptosis than their low-Mycn-expressing
counterparts. The order of administration signicantly inuenced antineo-
plastic efcacy, as exposure to AN-9 after radiation was more effective than
the reverse order. AN-9 and radiation exhibit additive antineoplastic effects
in neuroblastoma, a promising clinical approach, as these two therapies
have nonoverlapping toxicities. Although high-Mycn-expressing neuro-
blastoma cells were much more sensitive to radiation-induced apoptosis,
AN-9 inhibited Mycn expression. It follows that delivering radiation prior
to AN-9 would maximize the efcacy of combined HDACI and radiation
treatments. Combination therapy using HDACIs and radiation represents
an exciting new therapeutic approach to neuroblastoma.
102. A NOVEL THREE-DIMENSIONAL ALL HUMAN IN
VITRO BRAIN TUMOR INVASION MODEL
S.A. Murray,
1
H.K. Rooprai,
1
R. Selway,
2
and G.J. Pilkington
1
;
1
Cellular
& Molecular Neuro-oncology Group, School of Pharmacy & Biomedical
Sciences, University of Portsmouth, Portsmouth;
2
Department of
Neurosurgery, Kings College Hospital, Denmark Hill, London; UK
In order to study brain tumor invasion in vitro, previous three-dimensional
model systems have utilized pre-cultured chick heart fragment and rat
brain as targets in co-culture with human brain tumor. We have now suc-
ceeded in establishing a novel three-dimensional model whereby human
neoplastic glial cell spheroids are juxtaposed with spheroids derived from
non-neoplastic human brain tissue. Spheroids are developed from either
established glioma-derived cell lines or from primary cultures of glioma
biopsy as well as brain resected from patients who have undergone sur-
gery for epilepsy, short-term astrocyte-rich cell cultures derived therefrom.
Using the hanging drop method, 45,000 cells were inversely suspended
from a petri dish in 20 ml of DMEM. After either 24 h (fast-growing cells)
or 48 h (slower growing cells) in their gravity pools, spheroids are trans-
ferred to agar-coated petri dishes for a further 24 h prior to confrontation.
Cells were tracked with the use of uorescent cell trackers; progress was
recorded every 3 days for up to 15 days. Fluorescent cell trackers were also
employed for time-lapse video microscopy in the most invasive combina-
tion over 5 days. We are further developing the model in utilizing human
serum instead of fetal calf serum, which has been shown to alter antigenic
expression and growth rate of human glioma cells in two-dimensional cul-
tures within our laboratories. The effects of various agents that may impede
local brain tumor invasion are currently under investigation. To these ends
we are utilizing scanning and transmission electron microscopy, confocal
microscopy, total internal reected uorescence (TIRF) microscopy, and
live cell imaging utilizing Improvision Velocity and Openlab software on
a Zeiss inverted axiovert 200M microscope. This work was supported by
the Dr Hadwen Trust
103. AN IN VITRO DEMONSTRATION OF SYNERGISTIC
GLIOMA CELL KILL BY THE SELECTIVELY REPLICATION
COMPETENT ICP34. 5-NULL HSV MUTANT, HSV1716, AND
IONIZING RADIATION
S. Harrow,
1
R. Rampling,
1
A. Mace,
2
J. Plumb,
3
S. Brown,
2
and J.
Harland
2
;
1
Clinical Oncology,
2
Neurology,
3
Medical Oncology, The
University of Glasgow, Glasgow, Scotland, UK
Here we demonstrate, in vitro, in the human glioma cell line U373, a
synergistic relationship between cell kill from the oncolytic ICP34.5 null
herpes simplex virus mutant, HSV1716, and ionizing radiation. Additive
cell kill is shown in another human glioma cell line, MOG. Cell kill from
HSV1716 and ionizing radiation was investigated in vitro using the MTS
assay, a colorimetric method of determining the number of viable cells.
HSV1716 and ionizing radiation were combined to determine cell kill in the
human glioma MOG and U373 cell lines. Experiments combining HSV1716
and ionizing radiation indicated additional cell kill in U373 cells by day 6
compared to either modality in isolation. Isobologram analysis conrmed
a synergistic relationship between ionizing radiation and HSV1716 when
the U373 cells were irradiated one hour prior to virus inoculation. In the
MOG cell line the relationship in terms of cell kill appears to be additive.
Phase 1 studies in glioma patients have failed to demonstrate toxicity when
HSV1716 is injected into tumor or normal brain, nor when they proceeded
to receive further immunosuppressive treatment in the form of ionizing
radiation or chemotherapy. This supports the use of HSV1716 in conjunc-
tion with standard therapies but leaves open the question as to whether
combined therapy is likely to be additive or synergistic. Contradictory data
have been published on the possible synergy between ICP34.5 null HSV and
ionizing radiation in cell kill. In general, although the relationship between
the two modalities is dependent on factors such as the HSV mutant used,
the target cell type, and the scheduling of the virus and radiation treat-
ments, the in vivo data indicate that ICP34.5 null HSV and ionizing radia-
tion act synergistically in tumor cell kill. However, to our knowledge, there
has been no published data to date demonstrating synergy between the two
modalities in vitro. It has been proposed that the additional cell kill demon-
strated by various oncolytic HSV null mutants when combined with radia-
tion is due to the upregulation by radiation of certain cellular proteins such
as mammalian ribonucleotide reductase or GADD34, which complement
the missing gene in the attenuated virus. Multicycle growth experiments
showed that HSV1716 fails to replicate as efciently as wild-type HSV in
U373 cells. However, pre-irradiating U373 cells failed to result in enhanced
viral replication, suggesting that increased viral replication might not be the
reason for the increased cell kill. The demonstration of a synergistic rela-
tionship between ionizing radiation and HSV1716 in vitro is of interest if
these modalities are to be combined in clinical practice and justies further
investigation of the mechanisms involved.
104. PHYSICAL AND NUMERICAL MODELING
CEREBROSPINAL FLUID BEHAVIOR FOR DRUG DELIVERY
TO THE LEPTOMENINGES
D. Walker, S. Ammourah, A. Aroussi, M. Garnett, D. Giddings, L.
Howden, H. Power, and M. Vloeberghs; Childrens Brain Tumor
Research Centre, University of Nottingham, Nottingham, UK
Leptomeningeal dissemination (LMD) of tumor is a high risk in hema-
tological malignancies, primary brain tumors and parameningeal tumors
of childhood and adolescence, justifying either prophylactic or radical
CNS directed chemoradiotherapy. In leukemias, combined systemic and
intrathecal therapy has replaced cranial radiotherapy as prophylaxis; in
primary brain tumors, reducing doses of craniospinal or focal radiother-
apy in combination with systemic chemotherapy is being explored. In this
latter group, intrathecal therapy has not been extensively studied because
of uncertainties of drug selection and delivery. The purpose of this cross-
disciplinary study at the University of Nottingham is to reconsider the
design of intrathecal drug delivery methods for prevention of LMD. An
MRI scan of the entire CNS was obtained and data used to create 3D mod-
els of the ventricles and subarachnoid space (SAS). Work by Aroussi and
Vloeberghs has provided information about the ow in the cerebral aque-
duct and likely behavior of drugs injected into cerebral ventricles. Current
research is preparing increasingly realistic physical models of the CNS uid
ows, and CFD models using commercial engineering packages (Fluent).
The existing physical model is constructed from thin-walled acrylic sheet;
it contains the ventricles, a simplied brain surface, and spinal cord and the
boundary of the spine and skull. It is suspended in a transparent tank on a
mobile frame allowing rotation of the model and visual inspection of ow
tracers. The CFD model solves uid ow equations and can be programmed
to induce motion of the model boundaries, thus allowing investigation of
arterial pulsation of the brain and respiratory effects in the spine. For effec-
tive intrathecal delivery, the ow induced by CSF production alone is not
sufcient, and the ow emitted from the foramina of Lushka and Magendie
is transported mainly around the cranial SAS; diffusion is much less than
the likely convection of uid from the foramina to the arachnoid villi. Com-
putational work shows ow patterns that may be produced by pulsations
driven by physiology exterior to the SAS. Experimenting with models is
a relatively cheap technique for illuminating the likely behavior of highly
complex uid ow in this nearly inaccessible region of the human body.
The models will provide insight into current drug delivery behavior and
potential new delivery methods.
105. LACTACYSTIN EXHIBITS POTENT ANTITUMOR
ACTIVITY IN AN ANIMAL MODEL OF MALIGNANT
GLIOMA WHEN ADMINISTERED VIA CONTROLLED-
RELEASE POLYMERS
F. Legnani,
1
G. Pradilla,
2
Q. Thai,
2
B. Tyler,
2
G. Zamora,
1
S. Gaini,
3

F. DiMeco,
1
H. Brem,
2
and A. Olivi
2
;
1
Department of Neurosurgery,
Istituto Neurologico C. Besta, Milan, Italy;
2
Department of
Neurosurgery, Johns Hopkins University, Baltimore, Maryland, USA;
3
Universit degli Studi di Milano, Neurosurgery, Milan, Italy
Lactacystin, a proteasome-inhibitor, has been shown to induce apopto-
sis of experimental gliomas in vitro. However, its systemic toxicity prevents
further clinical use. To circumvent this problem, lactacystin can be deliv-
ered locally to the tumor site. We tested the efcacy of lactacystin incorpo-
rated into controlled-release polymers for treating experimental gliomas in
vitro and in vivo. 9L-Gliosarcoma and F98-glioma cell lines were treated
with lactacystin (10100 g/ml) for 72 h in vitro. Cell viability was mea-
sured with MTT assays. The toxicity of lactacystin/polycarboxyphenoxy-
propane-sebacic-acid (pCPP:SA) polymers was established in vivo by using
Fischer-344 rats that were intracranially implanted with lactacystin poly-
mers loaded from 0.1% up to 2% lactacystin by weight (w:w). The efcacy
of 1, 1.3, 1.5 and 1.7% lactacystin/pCPP:SA-polymers was determined in
Fischer-344 rats intracranially challenged with 9L and treated either simul-
taneously or 5 days after tumor implantation. Lactacystin was cytotoxic in
9L cells, causing a 16 8% ml that increased to 78 4% at 100 g/ml.
Similarly, lactacystin inhibited growth of F98 by 18 8% at 10 g/ml and
74 2% at 100 g/ml in vitro. Polymers released lactacystin for 21 days
and intracranial implantation in rats did not generate local nor systemic
toxicity at doses lower than 2% (w:w). Treatment with lactacystin/pCPP:
SA polymers with loading concentrations of 1.0, 1.3, and 1.5% prolonged
survival of animals intracranially challenged with 9L when polymers were
inserted in the day of tumor implantation. Lactacystin exhibits potent cyto-
toxic-activity against 9L and F98 in vitro. Furthermore, lactacystin can be
efciently incorporated and delivered using controlled-release polymers,
and at the proposed concentrations, lactacystin polymers are safe for CNS
delivery and prolong survival in the 9L model. These ndings support the
role of proteasome inhibitors in the treatment of malignant gliomas when
administered using local drug delivery.
106. GENETIC IDENTIFICATION OF GLIOBLASTOMA
MAINTAINING CELLS IN A XENOGRAFT MODEL
C. Glanz,
1
J. Rebetz,
1,2,4
Y. Stewnius,
3
D. Gisselsson,
3
A. Darabi,
1

E. Englund,
5
L.G. Salford,
1
B. Widegren,
1,2,4
and X. Fan
1,2,6
;
1
The
Rausing Laboratory, Division of Neurosurgery, Lund University
Hospital, Lund;
2
3
Department of Clinical
Genetics,
4
Division of Genetics, Department of Cell and Molecular
Biology,
5
Department of Pathology,
6
Section of Molecular Medicine and
Gene Therapy, Lund Strategic Research Center for Stem Cell Biology and
Cell Therapy, Lund University, Lund; Sweden
Glioblastoma (GBM) can consist of heterogeneous cell populations.
A small CD133 stem celllike population has been demonstrated to be
responsible for GBM maintenance. We have developed a serial transplant-
able xenograft GBM model to characterize the genetic background and
constitutive signaling pathways in GBM maintaining cells. Freshly isolated
GBM cells from a 47-year-old female patient at recurrence were subcuta-
neously injected into SCID-Beige mice. Xenograft GBM formed in initial
SCID-Beige mice were serially transplanted in SCID-Beige or NOD/SCID
mice without cell sorting for up to 4 passages. Freshly isolated GBM cells
and its xenograft cells at different passages were analyzed for cell surface
marker expression. Similar patterns of heterogeneous cell populations were
observed both in the GBM and its xenografts at all passages. CD44 and
epidermal growth factor receptor were expressed in 90% of the fresh GBM
and the xenograft GBM cells. The stem cell marker CD133 was expressed
in 70% of the primary GBM and about 30% of the xenograft GBM cells,
the platelet-derived growth factor receptor-a (PDGFRa) in 34% of the
primary GBM and about 20% of the xenograft GBM cells. However, the
immature neural ganglioside recognized by A2B5 was detected in 63%
of the primary GBM but not in the xenograft GBM cells. The same pat-
tern of heterogeneous cell populations was maintained in all passages of
xenograft GBM, suggesting that the GBM cell population hierarchy is to
a great extent maintained in this xenograft model and that a fraction of
GBM cells were capable of maintaining the xenograft GBM by self-renewal.
G-band karyotyping showed that the primary GBM cells exhibited great
intercellular heterogeneity with a wide variety of related subclones, all of
which showed complex karyotypes, including several whole-chromosome
losses and unbalanced translocations. In contrast, late passage xenograft
cells showed almost no intercellular variation and exhibited a complex
karyotype almost identical to one of the original GBM subclones, iden-
tied in 3 out of 25 cells in the primary GBM cells. This indicates that
most subclones in the primary GBM did not contribute to the maintenance
of xenograft GBM. Interestingly, the PDGFRa expression in the in vitro
cultured xenograft GBM cells was specically downregulated by a 6-day
cyclopamine treatment in a dose-dependent manner. Concomitantly, a
2.5-fold reduction of cell proliferation (P 0.01, n 3) was observed
at 10 mM cyclopamine treatment. Thus, constitutively active sonic hedge-
hog signaling critically contributes to GBM cell proliferation via PDGFRa
expression in this tumor system. Taken together, our data suggest that even
though GBMs consist of heterogeneous cell populations, only a small frac-
tion of these cells is responsible for tumor cell regeneration
107. COMPUTERIZED TIME-LAPSE MICROSCOPY OF
HUMAN GLIOMA INVASION IN ORGANOTYPIC BRAIN
SLICE CULTURES
B.W. Kristensen,
1
C. Bonde,
3
K. Eriksen,
1
C. Andersen,
2

H.D. Schrder,
1
and J. Zimmer
3
;
1
Institute of Pathology and
2
Department of Neurosurgery, Odense University Hospital, Odense;
3
Department of Anatomy and Neurobiology, University of Southern
Denmark, Odense; Denmark
The purpose of this study was to establish a computerized time-lapse
microscopy assay in order to monitor and characterize the growth of human
gliomas in organotypic brain slice cultures. Recent results have shown that
human glioma invasion in such cultures correlates with the histological
typing of the gliomas, suggesting that this model might be of consider-
able value in studying the mechanisms of brain tumor invasion. Human
glioma biopsy specimens were produced from freshly resected grade IIIV
gliomas and incubated with the uorescent dye DiI for live cell labeling.
Then the specimens were implanted in organotypic rat brain slice cultures
grown on semiporous membranes for 1 to 2 weeks according to standard
protocols. The slice cultures with implanted glioma biopsy specimens
(n 510 per case) were thereafter transferred for time-lapse uorescence
microscopy by using an automatic inverted uorescence microscope placed
in a specially constructed CO
2
incubator. Controlled by a computer pro-
gram developed locally by Bonde et al., the cultures were photographed by
uorescence and phase contrast microscopy every 30 min for up to 2 weeks,
only interrupted by change of culture medium. Larger series of slice cultures
with implanted glioma tissue (n 1236 per case) were photographed by
conventional inverted uorescence and phase contrast microscopy with
4- to 6-day intervals during 2 weeks. The results show that the invasion of
human glioma cells into organotypic brain slice cultures can be monitored
for up to 2 weeks by conventional and time-lapse microscopy in the present
setup, demonstrating how both glioma cell density and distance of inva-
sion increased with time. In contrast, when glioma biopsy specimens were
placed directly on semiporous membranes, no outgrowth of glioma cells on
the membrane was seen. The present study shows that computerized time-
lapse microscopy of uorescently labeled glioma cells implanted in rat brain
slice cultures provides a model for monitoring quantitative and qualita-
tive growth characteristics. This work was supported by the Danish MRC,
the Danish Cancer Research Foundation, Foundation in the memory of
Alice Bren, Grant in the memory of Einar Willumsen, Anniversary Grant
of King Christian IX and Queen Louise, King Christian X Foundation,
Simon Foughner Hartmanns Foundation and EU 5th FP (QLK3-CT-2001-
00407).
108. HUMAN GLIOBLASTOMA T98G CELLS
XENOGRAFTED IN ATHYMIC NUDE MICE
OVERWHELMINGLY COMMITTED APOPTOSIS AFTER
TREATMENT WITH ALL-TRANS-RETINOIC ACID AND
TAXOL
S. Ray,
1
S. Karmakar,
1
A. Das,
1
N. Banik,
1
and S. Patel
2
;
1
Departments
of Neurology and
2
Neurosurgery, Medical University of South Carolina,
Charleston, South Carolina, USA
Glioblastoma is the most prevalent and highly malignant brain tumor
in humans. It is not yet amenable to any currently available therapeutic
strategy and thus remains as a challenge both to basic scientists and to
physicians. Therefore, new therapeutic approaches need to be explored for
effective treatment of glioblastoma. We used all-trans-retinoic acid (ATRA)
and taxol (TXL) alone and also in combination for controlling the growth
of human malignant glioblastoma T98G cells xenografted in athymic nude
mice. For xenotransplantation of glioblastoma, 6 week-old athymic nude
mice (Charles Rivers) were subcutaneously (sc) injected with a (1:1) mix-
ture of exponentially growing T98G (6 million cells/mouse) and Matrigel.
Animals with 3-week-old glioblastoma xenografts were randomly divided
into 4 groups: control, ATRA, TXL, and ATRA plus TXL. Animals in the
control group did not receive any therapy. Each animal in the other groups
received intraperitoneally (ip) a daily dose of ATRA (1.5 g/kg), or TXL
(45 g/kg), or ATRA (1.5 g/kg) plus 4 h later TXL (45 g/kg) for 7 days.
Histopathological and biochemical experiments were conducted to evaluate
the efcacy of different treatments. Histopathological examination of H&E
stained tumor sections revealed that control tumors maintained character-
istic growth of glioblastoma, ATRA alone inhibited tumor cell proliferation
and caused astrocytic differentiation, TXL alone induced apoptosis to some
extent, and ATRA plus TXL caused signicant amounts of apoptosis in dif-
ferentiated cells. Differentiation of cells was associated with the downregu-
lation of telomerase activity. In situ immunouorescent labeling showed
calpain overexpression in apoptotic cells, suggesting a role for calpain in
mediation of apoptosis. Further, in situ TUNEL and double immunouo-
rescent labeling conrmed cell death with an increase in calpain expression
in tumor sections treated with TXL, or ATRA plus TXL. Western blot
analyses showed changes in expression of Bax and Bcl-2 proteins leading to
increased Bax:Bcl-2 ratio, cytochrome c release from mitochondria, activa-
tion of calpain and caspase-3 for degradation of 270 kD -spectrin at the
specic sites to generate 145 kD spectrin breakdown product (SBDP) and
120 kD SBDP, respectively, in the course of apoptosis. Our investigation
in an animal model revealed that treatment of xenografted glioblastoma
with ATRA plus TXL induced differentiation and apoptosis for controlling
malignant growth.
109. INTERACTIONS OF NEURAL STEM CELLS
AND GLIOMA CELLS IN A THREE-DIMENSIONAL
ORGANOTYPIC BRAIN SLICE CO- CULTURE SYSTEM
O. Heese, D. Zirkel, M. Westphal, and K. Lamszus; University Hospital
Hamburg-Eppendorf, Neurosurgery, Hamburg, Germany
Various in vivo studies have demonstrated a migration tendency of
neural stem cells (NSCs) toward intracranial gliomas, making these cells a
potential carrier for delivery of therapeutic genes to disseminated gliomas.
We analyzed NSC migration in response to glioma stimuli in an organo-
typic brain slice model in order to mimic the in vivo microenvironment
including the three-dimensional architecture of murine brain tissue. The
chemotactic effects of 5 glioma cell lines and their responding conditioned
media on the murine NSC line C17.2 in a co-culture organotypic brain
slice system were assessed. NSCs and glioma cells were identied inside
the standardized murine brain slice by pre-implantation staining with DiI
and DiO. Migration of NSCs and glioma cells inside the brain slices was
characterized and quantied by using a confocal laser microscope on days
2, 6, and 12. C17.2 NSCs migrate inside the brain slices and seem to fol-
low preserved anatomic structures. Migration of the NSCs was modied
by conditioned media of glioma cells. Conditioned media of two glioma
cell lines augmented migration of NSCs up to 50% compared to controls.
In two gliomas, conditioned media stimulation was only moderate (20%).
Conditioned media of one cell line produced inhibition of NSC migration.
Co-culturing of NSCs and glioma cells inside the brain slice resulted in a
directed migration of both cell types toward each other in 3 of 5 glioma cell
lines. The organotypic brain slice model displays several advantages over
less complex in vitro migration models since a physiologic microenviron-
ment of brain tissue and a three-dimensional architecture are preserved.
Migration of NSCs toward gliomas in our assay system seems to depend
on individual phenotypic characteristics and growth factor release patterns
of the target tissue.
110. INTERSTITIAL PHOTODYNAMIC THERAPY OF
RECURRENT MALIGNANT GLIOMAS USING
5-AMINOLEVULINIC ACID (5-ALA)
J. Mehrkens,
1
W. Stummer,
3
T. Beck,
2
J. Tonn,
1
and F. Kreth
1
;
1
Department of Neurosurgery and
2
Laser-Research-Laboratory,
Ludwig-Maximilians-Universitt, Mnchen;
3
Department of
Neurosurgery, Heinrich-Heine-University, Dusseldorf; Germany
Photodynamic therapy (PDT) might have the potential to improve
local tumor control in selected patients: In PDT a photosensitizer (PS) is
transferred selectively in tumor cells and activated by light of an appropri-
ate wavelength, which leads to cytotoxic reactions. However, uncertain-
ties concerning dosimetry and PS-distribution as well as therapy-related
side effects have limited the clinical impact of PDT. These disadvantages
might be overcome by the concept of stereotactic interstitial PDT (iPDT)
using 5-ALA as PS. iPDT was considered to be indicated for patients with
a minimum Karnofsky performance status (KPS) of 70 with a circum-
scribed recurrence of a malignant glioma after prior multimodal therapy
with a maximum diameter of 3 cm. All operations were performed under
general anesthesia. Patients received 20 mg/kg 5-ALA 1 h preoperatively.
After tumor histology had been veried by stereotactic biopsy, 3D-multi-
modal-treatmentplanning (CT, MRT, FET-PET) followed by stereotactic
implantation of up to 6 laser-probes was performed. Irradiation time was
60 min (Ceralas PDT Diode Laser: wavelength 633 nm, power 200 mW/cm
[BioLitec AG, Jena, Germany]). Therapy effects were documented by MRI
(T1, T2 contrast) 24 h, 4 weeks, and then in 3 month-intervals postopera-
tively. Between October 2002 and December 2003, 10 adult patients (mean
age 54, range 3172 years; mean tumor volume 7.9 ml, range 2.126 ml)
were included. The applied volume-dose was in the range of 1000 to 1500
J/cm
3
. Early MRI follow-up within 24 h post-treatment showed a com-
plete resolution of the contrast-enhanced lesion in 7 patients and a partial
response in the other 3 patients. There was no enhanced treatmentinduced
brain edema. Thus, steroids were only applied for 3 days, as routinely done
in other stereotactic procedures in our institution. The estimated 1-year
survival rate was 70% (3 patients had died on last follow-up). There was
no surgery- or treatment-related morbidity or mortality. Stereotactic iPDT
using 5-ALA is a minimally invasive and low-risk therapy. The multimodal
3-D treatment planning for the rst time allows for an exact three-dimen-
sional dosimetry and irradiation of a dened tumor volume.
111. HSV1716 INJECTION INTO BRAIN ADJACENT TO
TUMOR FOLLOWING SURGICAL RESECTION OF
HIGH-GRADE GLIOMA: SAFETY DATA AND LONG-TERM
SURVIVAL
S. Harrow,
1
V. Papanastassiou,
2
J. Harland,
3
R. Mabbs,
5
R. Petty,
3

M. Fraser,
2
D. Hadley,
4
J. Patterson,
6
S. Brown,
5
and R. Rampling
1
;
1
Beatson Oncology Centre, Clinical Oncology, and Departments of
2
Neurosurgery,
3
Neurology, and
4
Neuroradiology, The University of
Glasgow, Glasgow, Scotland;
5
Cruscade Laboratories Ltd., Glasgow,
Scotland,
6
Clinical Physics, South Glasgow University Hospitals,
Glasgow, Scotland; UK
In this study the authors have demonstrated that herpes simples virus
1716 (HSV1716) is safe following injection into brain adjacent to excised
high-grade glioma, in patients who proceeded to receive further immuno-
suppressive radiotherapy or chemotherapy. Twelve patients, six with recur-
rent disease and six with newly diagnosed high-grade glioma, underwent
maximal resection of the tumor. Following resection, 1 ml of 10
5
pfu of
HSV1716 was injected into the brain adjacent to tumor. Aliquots of 0.1
to 0.2 ml of HSV1716, were injected into eight to ten sites of the brain
adjacent to tumor with the intent of infecting residual tumor cells. Patients
were reviewed daily in the rst week, weekly for six weeks, then twice
monthly for the rst year, and then on a six-month basis. Clinical and neu-
rological parameters were recorded and blood samples were obtained for
hematological, biochemical, and serological assessment. In addition, MRI,
or CT if MRI was not tolerated, with and without contrast, was performed
pre- and postoperatively, and then in line with the clinical assessment
review schedule. Patients also underwent SPECT (single photon emission
computed tomography) with thallium-201 used to identify areas of high
cellular metabolic activity reecting high-grade tumor growth. Patients
underwent imaging outwith protocol when clinically indicated. There was
no clinical evidence of toxicity during the period of formal follow-up or
during the period associated with the administration of HSV1716. Longi-
tudinal follow-up, until February 2003, has allowed assessment of overall
survival compared to that of similar patients not treated with HSV1716.
Three patients remain alive and clinically stable at 15, 18, and 22 months
postsurgery and HSV1716 injection. Remarkably, the rst patient in the
trial, who had extensive recurrent disease pre-procedure, was alive at 22
months following injection of HSV1716 and 29 months following rst
diagnosis. Imaging demonstrated a reduction of residual tumor over the
22-month period despite no further medical intervention since surgery and
HSV1716 injection. In this study we demonstrate that on the basis of clini-
cal observations, there has been no toxicity following the administration of
HSV1716 into the resection cavity rim in patients with high-grade glioma.
The survival and imaging data, in addition to the lack of toxicity, give us
condence to proceed to a clinical trial to demonstrate efcacy of HSV1716
in glioma patients.
112. TEMOZOLOMIDE/PEGYLATED LIPOSOMAL
DOXORUBICIN (PLD) IN PROGRESSIVE GLIOBLASTOMA
MULTIFORME (GBM)
G. Dresemann; Onkologische Abteilung, Franz-Hospital Duelmen,
Duelmen, Germany
GBM still is one of the most aggressive malignancies, with a median
survival of 1 year. Two-year progression-free survival is about 11% in
newly diagnosed cases, and 5-year overall survival less than 5%, so most
of the patients (pts) will experience a progression within the rst 2 years.
Although magnetic resonance imaging (MRI) seems to indicate deciency
in the blood-brain barrier (BBB), only drugs penetrating the intact BBB
have in vivo efcacy in GBM, with a median survival benet of about 3
months only. Temozolomide (T) is one the most effective drugs in progres-
sive GBM. Long-term survival, however, remains limited. Doxorubicin,
a very effective drug in GBM in vitro, does not penetrate the BBB and
therefore did not show in vivo effectivity. Pegylated liposomal doxorubi-
cin (PLD) is able to penetrate the BBB and has shown modest activity in
temozolomide refractory GBM. The drug proles of T and PLD highly sug-
gest additive activity without overlapping toxicity. From August 2001 to
May 2004, 40 pretreated (surgery, irradiation therapy, 1 chemotherapeutic
regimen) GBM pts were treated with T/PLD for progression. T was given
on days 15, 812, 1519 dosed 200 mg per day for pts up to 70 kg of
bodyweight and 250 mg per day for pts over 70 kg, combined with PLD 30
mg/m body surface on day 1 intravenously to be repeated every 4 weeks.
Prolonged dosage of T was chosen because of enzyme induction with the
potency of increased efcacy. Blood cell counts were taken weekly, and
MRI scans were performed every 8 weeks for disease monitoring. T/PLD
caused III hematotoxicity in 6/40 pts II hematotoxicity in 16/40 pts, there
were no febrile neutropenias, 4/40 pts had III nausea/vomiting requiring
5HT3 antagonists. One of 40 pts had a complete response lasting for 33
months, 4/40 had a partial response with a median duration of 23 months
(825), 24 pts had a stable disease with a median duration of 6 months
(315), and 11 pts were primary progressive. Median progression-free sur-
vival (PFS) was 5 months, median overall survival (OS) 7 months, PFS after
12 months was 6/40 pts, and OS at 12 months was 9/40 pts. T/PGD was
well tolerated and effective in this group of 40 progressive pretreated GBM
pts. These data should be evaluated by further clinical and pharmacologi-
cal examination.
113. CONVECTION-ENHANCED DELIVERY OF PACLITAXEL
IN PATIENTS WITH RECURRENT GLIOBLASTOMA
MULTIFORME: A PHASE 1/2 STUDY
R. Goldbrunner,
1
P. Tanner,
1
M. Holtmannspoetter,
2
G. Poepperl,
3

W. Rachinger,
1
F. Kreth,
1
and J. Tonn
1
; Departments of
1
Neurosurgery,
2
Neuroradiology, and
3
Nuclear Medicine, Universiteitsklinikum
Grosshadern, Munchen, Germany
Critical issues in chemotherapy of malignant gliomas are systemic tox-
icity and the ability of bypassing the blood-brain barrier. To overcome these
issues, direct drug delivery into the brain parenchyma is increasingly used.
Utilizing slight pressure gradients, convection can be used to achieve a dis-
tribution volume sufcient to cover the brain tumor and the surrounding
tissue. Effective dose, efcacy, and safety of convection enhanced deliv-
ery (CED) of paclitaxel (Taxol) in patients with recurrent glioblastoma
multiforme (GBM) are evaluated in a phase 1/2 trial. Eight patients with
recurrent GBM were enrolled in this study each completing up to 2 cycles
of intratumoral paclitaxel infusion. One to two catheters were placed ste-
reotactically into the solid tumor on the basis of MRI and FET-PET data.
Paclitaxel dissolved in Cremophore at a concentration of 0.25 to 0.5 mg/ml
was infused in a rate of 0.3 ml/h for 5 days, resulting in a total dose of 9 to
18 mg paclitaxel. Convection was monitored by MRI including DWI and
DTI. Follow-up including MRI and FET-PET was performed at day 28,
then bimonthly for 1 year. Tumors were located within or next to speech or
central areas in 7/8 patients. Initial MRI studies demonstrated induction of
intratumoral necrosis in 7/8 patients. Partial response (as shown by MRI
and PET) was seen in 7/8 patients, stable disease in 1/8. Median progres-
sion-free survival was 7.0 months, and median overall survival was 10.0
months (range, 415 months). Permanent neurological decit was seen in 2
patients, temporary decits during paclitaxel infusion in 4/8. There were no
signs of any systemic toxicity. According to these preliminary results, CED
of paclitaxel is a very effective therapy for patients with recurrent glioblas-
toma multiforme with an acceptable safety prole. Long-term observations
with a larger number of patients will follow.
114. PERITUMORAL CONVECTION-ENHANCED
DELIVERY (CED) OF IL13-PE38QQR (IL13PE): RESULTS OF
MULTICENTER PHASE 1 STUDIES IN RECURRENT HIGH
GRADE GLIOMA (HGG)
S. Kunwar,
1
Z. Ram,
2
J.H. Sampson,
3
M. Prados,
1
S. Chang,
1
F.F. Lang,
4

J.M. Piepmeier,
5
P.H. Gutin,
6
D. Croteau,
7
and R.K. Puri
8
;
1
University
of California, San Francisco, California, USA;
2
Tel Aviv University,
Tel Aviv, Israel;
3
Duke University Medical Center, Durham, North
Carolina, USA;
4
M.D. Anderson Cancer Center, Houston, Texas, USA;
5
Yale University School of Medicine, New Haven, Connecticut, USA;
6
Memorial Sloan-Kettering Cancer Center, New York City, New York,
USA;
7
NeoPharm, Inc., Lake Forest, USA;
8
CBER, U.S. FDA, Bethesda,
Maryland, USA
IL13PE is a recombinant protein consisting of IL13 and truncated Pseu-
domonas exotoxin that has selective antitumor activity against malignant
glioma cells overexpressing the IL13 receptor. We assessed the safety and
efcacy of peritumoral CED of IL13PE in patients with recurrent HGG.
Three Phase 1 studies evaluated peritumoral CED of IL13PE following
tumor resection. Eligibility criteria included: age 18, KPS 70, and a
tumor amenable to resection. The rst study (002) determined the maxi-
mum tolerated infusate concentration (MTD) of IL13PE, using a dose esca-
lation design. Infusion duration and feasibility and accuracy of postresec-
tion catheter positioning were also investigated. A second study (103R02)
expanded the patient population treated at the MTD, and a third study
(105) further assessed drug distribution at the MTD. Patients underwent
tumor resection followed by a single 46 day infusion of 0.251.0 g/ml of
IL13PE (total dose 1872 g) and were followed with serial MRI scans.
Enrollment is complete; 51 patients received study drug via peritumoral
infusion. Forty-ve patients had histologically conrmed recurrent glioblas-
toma multiforme (GBM: study 002, n 38; 103R02, n 3; 105, n 4).
Maximum tolerated infusate concentration was 0.5 g/ml. Most frequent
adverse events (AEs) were headaches and hemiparesis. There were 2 grade
4 toxicities at the highest concentration, and 9 grade 3 AEs reported in the
study group. Concentration-dependent imaging changes related to study
drug were also observed. Median overall survival (OS) for GBM patients
was 45.9 weeks. Median OS for GBM patients with peritumoral infusate
concentration of 0.5 g/ml (n 24) is currently 70.3 weeks. Eight patients
remain progression free (24 to 174 weeks, median 69 weeks). Ini-
tially, 50% of intraoperatively placed catheters were positioned accurately;
accuracy increased to 90% when postresection stereotaxis was utilized.
IL13PE appears to have a favorable risk-benet prole, and prolonged over-
all survival has been observed. Postresection catheter placement appears to
enhance positioning accuracy and, potentially, drug distribution and OS.
Design of the ongoing international phase 3 trial to determine efcacy and
safety of IL13PE in rst relapse GBM is based on these ndings.
115. INDIVIDUALIZED CHEMOTHERAPY FOR MALIGNANT
ASTROCYTOMAS BASED ON O6-METHYLGUANINE-DNA
METHYLTRANSFERASE METHYLATION ANALYSIS
T. Watanabe, A. Ogino, T. Ohta, A. Yoshino, and Y. Katayama; Nihon
University School of Medicine, Neurological Surgery, Tokyo, Japan
The correlation between O6-methylguanine-DNA methyltransferase
(MGMT) methylation and responsiveness to nitrosourea chemotherapy
observed in recent clinical studies suggests that use of this alkylating agent
should be reserved for MGMT-methylated tumors. MGMT appears not
to be linked to platinum resistance, which makes platinum chemotherapy
a good candidate for the treatment of MGMT-unmethylated tumors. We
instituted a preliminary trial of individualized chemotherapy based on
MGMT methylation status combining interferon and radiation therapy for
patients with malignant astrocytomas. A total of 20 patients with newly
diagnosed malignant astrocytomas were enrolled onto the study. Seven
patients with MGMT-methylated tumors were treated with the procar-
bazine, 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-2(2-chloroethyl)-3-
nitrosourea (ACNU), and vincristine (PAV) regimen. An objective response
to the PAV therapy was noted in all 3 patients with measurable residual
tumor (2 complete responses and one partial response). Five patients con-
tinued to be disease free as of 10, 17, 19, 22, and 26 months after initiation
of the PAV therapy. The 13 patients with MGMT-unmethylated tumors
were treated with the carboplatin and etoposide (CE) regimen. Only one
(14%) of 7 patients with assessable disease partially responded to the
CE therapy. Three patients were free from progression at 11, 14, and 20
months, whereas the remaining 10 patients progressed early following com-
mencement of the CE administration. Our results provide support for the
responsiveness of MGMT-methylated malignant astrocytomas to the PAV
regimen, but do not justify the usefulness for the CE regimen in unmethyl-
ated tumors.
116. A PHASE 1/2 TRIAL OF TEMOZOLOMIDE AND
VINORELBINE IN PATIENTS WITH RECURRENT BRAIN
METASTASES
A.M.P. Omuro, J.J. Raizer, and L.E. Abrey; Memorial Sloan-Kettering
Cancer Center, Department of Neurology, New York, New York, USA
Treatment options for recurrent brain metastasis (BM) are limited.
Temozolomide (TMZ) is an alkylating agent with efcacy in BM. Vinorel-
bine is a lipophilic agent with efcacy in a variety of solid tumors and can
potentially cross the blood-brain barrier. We designed a phase 1/2 trial for
recurrent BM utilizing vinorelbine in combination with TMZ in a pro-
tracted schedule. Patients with solid tumors and recurrent or progressive
BM were eligible. One cycle was dened as 28 days. TMZ was given on
a dose of 150 mg/m
2
on days 1 to 7 and 15 to 21. Vinorelbine was given
on days 1 and 8 at escalating doses for the phase 1, with a starting dose of
15 mg/m
2
and increments of 5 mg/m
2
for each cohort of 3 patients, until
maximum tolerated dose (MTD) or a dose of 30 mg/m
2
was reached. For
the phase 2, 20 pts would be treated at the MTD; if two or more major
responses were seen, sample size would be increased to 35. Twenty-seven
pts have been enrolled (phase 1, 18 pts; phase 2, 9 pts). Median age was 60
(4076); 11 pts were men, median KPS was 80 (70100). Primary cancer
was lung in 16 pts (non-small cell, 13 pts), breast in 8, renal in 1, endome-
trial in 1. During the phase 1, no dose-limiting toxicity was observed and
the phase 2 dose of vinorelbine has been 30 mg/m
2
. Myelotoxicity has been
the main adverse event, with grades 3 or 4 neutropenia in 7 pts, lymphope-
nia in 14, anemia in 2, and thrombocytopenia in 3. Response has been eval-
uated in 18 pts (complete response, 1; partial response, 1; minor response,1;
stable disease, 5; progressive disease, 10). Median time to progression was
2 (16) months. This regimen is well tolerated, and preliminary results
suggest promising efcacy. The phase 2 sample size will be increased to 35
pts. Updated results will be presented.
117. AN UPDATE OF PHASE 2 TRIAL RESULTS: PATIENTS
WITH RECURRENT MALIGNANT GLIOMA TREATED
WITH IODINE 131-LABELED MURINE ANTITENASCIN
MONOCLONAL ANTIBODY 81C6 INTO THE RESECTION
CAVITY
D. Reardon, G. Akabani, A. Friedman, H. Friedman, R. McLendon,
J. Quinn, J. Rich, J. Vredenburgh, M. Zalutsky, and D. Bigner; Duke
University Medical Center, Durham, North Carolina, USA
We previously established the maximum tolerated dose of iodine 131-
labeled murine antitenascin antibody 81C6 (
131
I-81C6) administered into
a surgically created resection cavity (SCRC) for the treatment of recur-
rent malignant glioma in adult patients to be 100 mCi. The current phase
2 study is designed to evaluate the clinical activity of a 100-mCi dose of
131
I-81C6 administered to patients with recurrent malignant glioma and to
further evaluate the safety and toxicity of this approach. Eligibility criteria
included the following: adults with recurrent malignant CNS tumor; gross
total resection; lack of communication between the resection cavity and the
CSF space; KPS greater than 60%; and adequate bone marrow, hepatic, and
renal function. To date 43 patients have been enrolled, including 34 with
either GBM or gliosarcoma, 6 with AA, 2 with AO, and 1 with metastatic
adenocarcinoma. Ninety-three percent received prior external beam radia-
tion, and 51% were treated with prior chemotherapy. The median age was
54.5 years (range, 2677) and 63% were males. All patients received 100
mCi except for two patients who received 67 mCi and 75 mCi respectively
because of the limited size of the SCRC. Reversible hematologic toxicity
occurred in 27% of patients, and 5 patients (15%) developed delayed neu-
rotoxicity. However, only one patient required reoperation for symptomatic
radionecrosis. The median survival of all patients and of those with GBM
was 67.5 and 63.2 weeks, respectively. For patients with GBM the prob-
ability of 1-year survival is 0.56 (95% CI, 0.410.78). These encouraging
results suggest that further study of
131
I-81C6 for patients with recurrent
CNS tumors is warranted.
118. PROGNOSTIC RELEVANCE OF CLINICAL AND
THERAPEUTICAL ASPECTS IN PATIENTS WITH
GLIOBLASTOMA MULTIFORME. ANALYSIS OF
99 PATIENTS
A. Mangiola,
1
C. Anile,
1
P. De Bonis,
1
B. Pettorini,
1
G. Sabatino,
1
S.
Mannino,
1
C. Pedone,
2
and G. Maira
1
;
1
Neurosurgery, Neurological
Sciences;
2
Geriatrics Gerontological, Geriatrics and Physiatric Sciences,
Catholic University, Rome, Italy
Glioblastoma multiforme (GBM) is the most common and lethal pri-
mary brain tumor in adults. Despite advances in research and treatment, the
prognosis for these patients remains poor, with a median survival of 1 year.
Current management strategies used for GBM include surgery followed by
adjunctive radiation therapy and chemotherapy. There is controversy among
the neurosurgeons regarding surgical strategies. The purpose of this study
was to identify clinical and therapeutic predictors of survival. We retrospec-
tively analyzed 99 consecutive patients with primary supratentorial GBM
who underwent tumor removal at our institution between 1999 and 2003.
After surgery patients were treated with adjuvant therapy: radiotherapy,
chemotherapy, radioimmunotherapy. Of 99, 23 underwent a reinterven-
tion after recurrence. Based on different therapeutic strategies and clinical
aspects different subgroups have been dened. We determined the median
survival of each one using the Kaplan-Maier method. In concordance with
literature we have obtained that age and total removal are predictors of
survival. Other data differ from literature: survival according to Karnofsky
Performance Scale score, epilepsy as rst symptom, and functional location
and presence of central necrosis were not statistically signicant. The most
interesting results come from therapeutics. Patients who underwent total
en bloc resection had a survival of 19 months, while total inside-out resec-
tion group had a survival of 12 months (P 0.0005). Patients who under-
went one surgical treatment had a survival of 10.5 months, and patients
who underwent more surgical treatments had a survival of 22 months P
0.002). Survival according to adjuvant therapy has been 23 months (radio-
therapy, chemotherapy, radioimmunotherapy), 18 months (radiotherapy,
chemotherapy), and 14 months (radiotherapy) (P 0.002). In our series
the en bloc resection represents an important prognostic variable. When
tumor recurs, a re-intervention has to be considered. The treatment of GBM
remains multidisciplinary. In our experience the best adjuvant treatment is
radio chemo radioimmunotherapy.
119. PHASE 1 STUDY RESULTS OF GEFITINIB (IRESSA;
ZD1839) PLUS RAPAMYCIN IN THE TREATMENT OF
PATIENTS WITH RECURRENT MALIGNANT GLIOMA
D.A. Reardon, J. Rich, J. Quinn, J. Vredenburgh, A. Desjardins, S.
Sathornsumetee, S. Gururangan, P. Lyons, D. Bigner, and H. Friedman;
Duke University, Durham, North Carolina, USA
Aberrant signaling of the PI3K/Akt pathway is common in malignant
glioma. The primary objective of this phase 1 study is to determine the max-
imum tolerated dose (MTD) and dose-limiting toxicity (DLT) of Getinib,
an inhibitor of the EGFR receptor tyrosine kinase, when combined with
rapamycin, a macrolide antibiotic capable of inhibiting mTOR, a critical
downstream regulator of PI3K/Akt signaling, among patients with recur-
rent malignant glioma. Eligibility criteria include the following: recurrent
malignant glioma; fewer than 4 prior episodes of recurrence; greater than
18 years of age; KPS greater than 60%; adequate hepatic, renal, pulmo-
nary and bone marrow function. Patients previously treated with EGFR
or mTOR-directed therapies are excluded. Patients are stratied based on
concurrent use of enzyme-inducing anticonvulsants (EIAC; phenytoin, car-
bamazepine, and phenobarbital). A standard 33 dose escalation design
was employed with both strata independently escalated. Gefitinib and
rapamycin are administered on a continuous daily dosing regimen. Each
treatment cycle is 28 days, and patients are evaluated for response every
other cycle. Twenty-three patients have been enrolled to date, including 20
with recurrent GBM and 3 with recurrent AA. The median age is 51 (range,
3366); 65% are male, and 52% are on EIAC. Accrual and dose escalation
are ongoing, and the MTD has yet to be dened for either stratum. The
only DLT to date was an episode of grade 3 mucositis. Pharmacokinetic
sampling has been collected in approximately half of patients. Six patients
have discontinued therapy due to progressive disease, while 17 continue on
study having received 1 to 3 cycles of therapy. One marked radiographic
response has been observed to date. An update of the outcome and toxicity
of Getinib plus rapamycin for patients with recurrent malignant glioma
will be presented based on additional follow-up and enrollment.
120. ENHANCED DELIVERY OF CHEMOTHERAPY BY
OSMOTIC BLOOD-BRAIN BARRIER DISRUPTION (BBBD)
WITH DEFERRED RADIOTHERAPY FOR TREATMENT
OF PRIMARY CNS LYMPHOMA (PCNSL): ANALYSIS OF 33
PATIENTS
T. Siegal,
1
E. Shalom,
1
E. Linetsky,
1
A. Taliansky,
1
A. Lossos,
1
F.
Bokstein,
1
N. Levin,
1
A. Schwartz,
2
and J. Gomori
2
;
1
Leslie and Michael
Gafn Center for Neuro-Oncology;
2
Neuro-Radiology Unit, Hadassah
Hebrew University Hospital, Jerusalem, Israel
The optimal therapy for PCNSL has not yet been established. The
addition of high-dose chemotherapy (chTx) to radiotherapy substantially
improves median survival, but the combined therapy carries a high risk
of severe treatment-induced delayed neurotoxicity. Several studies tried to
use intensive chTx as the sole treatment for PCNSL but their limitations
proved to be either a low response rate, short duration of response, or a high
rate of treatment-related death (10%). A single institutions experience with
delivery of chTx in conjunction with osmotic BBBD, without subsequent
radiotherapy, was reported to yield a survival rate similar to that observed
in standard regimens that use both chTx and radiation therapy. This BBBD-
enhanced therapy was not associated with delayed neurotoxicity. We report
the experience at Hadassah University Hospital with BBBD-enhanced chTx
without radiotherapy in PCNSL. Pts with non-AIDS related PCNSL were
prospectively treated with methotrexate-based chTx as rst-line treatment
or with carboplatin-based therapy if they failed any previous methotrexate
treatment. Both regimens were given in conjunction with BBBD and with
no subsequent radiotherapy. We treated 33 pts whose median age was 48
(range, 2368). CSF spread and intraocular lymphoma were each present
in 12% of the pts. The mean number of treatment cycles/pt was 7, with
15% of cycles based on carboplatin with a total of 474 BBBD procedures.
A complete response was achieved in 23 pts (70%). Median follow-up is
27 months (range, 594), and the median survival is 39 months with a
5-year survival rate of 41%. Three pts died of causes other than disease
progression; one was treatment related. Other procedure-related complica-
tions included 2 minor strokes, 2 arterial injuries successfully repaired by
stenting, and one reversible brainstem lesion induced by carboplatin. The
median time to tumor progression has not yet been reached (13/33 pro-
gressed) with 24- and 36-month PFS rates of 69% and 57%, respectively.
Of pts followed for more than 24 months, none demonstrated delayed leu-
koencephalopathy, and there was no progressive cognitive loss on clinical
and neuropsychological testing. BBBD-enhanced chTx without subsequent
radiotherapy requires the expertise of a multidisciplinary team to treat pts
with PCNSL. It results in PFS and survival outcomes similar to those with
standard therapy that use radiotherapy, but offers a favorable long-term
cognitive outcome.
121. TEMOZOLOMIDE (TMZ) FOR 21/28 DAYS IN
PATIENTS WITH PROGRESSIVE GLIOBLASTOMA (GBM):
PRELIMINARY RESULTS OF PHASE 2 STUDY OF GICNO
(ITALIAN NEURO-ONCOLOGY GROUP)
A. Brandes,
1
G. Cavallo,
2
A. Tosoni,
1
M. Ermani,
3
L. Scopece,
2
E.
Franceschi,
2
L. Nicolardi,
1
A. Talacchi,
4
V. Blatt,
1
and L. Crin
2
;
1
Medical Oncology, Ospedale Busonera, Padova;
2
Department
of Medical Oncology, Ospedale Bellaria, Bologna;
3
Department
of Neurological Sciences, Azienda Ospedale Universit, Padova;
4
Departmentt of Neurosurgery, University Hospital, Verona; Italy
The activity of TMZ, at a standard schedule of 200 mg/m
2
for ve
days every 28 days, appears encouraging in patients (pts) with relapsed
and newly diagnosed malignant gliomas. A continuous dose schedule of
TMZ leads to DNA repair enzyme AGAT level depletion in tumor cells.
AGAT, by removing TMZ-produced methyl adducts, contributes to resis-
tance to alkylating agents. The aim of the present study was therefore to
determine the efcacy and safety of continuous low-dose TMZ adminis-
tration in patients with GBM. Twenty pts (11 males, 9 females; median
age, 56 years [range, 3171]) with GBM progressive/recurrent after surgery
and radiotherapy received oral TMZ 75 mg/m
2
for 21 consecutive days,
every 28 days. All pts were chemo-nave. Preliminary data on efcacy and
safety were available for 20 pts. PFS-6 was 34% (95% CI, 18%66%),
and median TTP was 15 weeks (95% CI, 12.4NA). A total of 80 cycles
were delivered (mean 4 per pt; range, 18). Grade 2 and 3 lymphopenia was
observed in 15% and 30% of pts, respectively. Neutropenia was G3 in 1
patient (5%) and G4 in 1 (5%). Hepatic G1 and G3 toxicity was observed
in 15% and 5% of patients respectively. The TMZ dose was reduced to
75% in one cycle (1.2%) because G3 neutropenia and G3 lymphopenia
occurred. This regimen appears to be well tolerated and active in patients
with progressive GBM.
122. MODERATE HEMATOLOGICAL TOXICITY OF DOSE-
INTENSIFIED TEMOZOLOMIDE (TMZ) IN PATIENTS WITH
RECURRENT GLIOBLASTOMA
W. Wick and W. Weller; Department of General Neurology and
Hertie Institute for Clinical Brain Research, University of Tuebingen,
Tuebingen, Germany
A one-week-on/one-week-off TMZ schedule had shown clinical ef-
cacy and moderate hematological toxicity in 21 patients with recurrent
glioblastoma. In a reassessment of our database, 39 patients with recurrent
or progressive glioblastoma treated with the one-week-on/one-week-off
regimen of TMZ, starting at 150 mg/m
2
with dose adaptation in 25 mg/m
2

steps, have been evaluated according to leukocyte and platelet counts which
were determined in weekly intervals. The analysis similarly to the published
data demonstrated a promising progression-free survival rate of 43% at 6
months. The survival rate at 12 months from recurrence was 21%. Hema-
tological toxicity in a total of 382 treatment weeks was low. Thirty-two of
39 patients never experienced leukocyte counts below 3,000/l or platelet
counts below 100,000/l. Thrombopenia necessitated platelet transfusions
in 5 patients. Two patients experienced grade 3/4 hematological toxicity
according to the common terminology criteria for adverse events later than
3 months after they started on TMZ. All other patients suffered such toxic-
ity within the rst weeks of TMZ. None of the 13 patients receiving TMZ
for more than 6 months experienced grade 3/4 hematological toxicity. Our
present extended analysis of the continuous one week on/one week off
schedule of TMZ conrms the safety and efcacy of this regimen in recur-
rent or progressive glioblastoma.
123. LOOK FOR ANXIETY IN THE YOUNG AND
DEPRESSION IN THE PREVIOUSLY DEPRESSED IN
POSTOPERATIVE PATIENTS WITH BRAIN TUMORS
L. Kilbride,
1
and R. Grant
2
;
1
School of Acute & Continuing Care
Nursing, Napier University, Edinburgh, Scotland;
2
Edinburgh Centre for
Neuro Oncology, Lothian University NHS Trust, Edinburgh, Scotland;
UK
Anxiety and depression can be symptoms that limit the quality of life of
patients if not adequately diagnosed or treated. The period between surgery
and radiotherapy for patients with brain tumors is a period where anxiety
and depression commonly occur. We prospectively studied patients at 3
time points after surgery to study the frequency of anxiety and depres-
sion, the most likely time point for developing symptoms and any features
that might act as a predictor of anxiety or depression. Fifty-one consecu-
tive patients with intrinsic brain tumors gave consent to be involved in the
study. Only 34/51 completed the Hospital Anxiety and Depression (HAD)
scale at three points. A full history was taken, and a HAD scale score was
obtained postsurgery, three weeks postsurgery, and pre-radiotherapy. A
HAD score of 11 was considered abnormal. Thirty-eight patients (74%)
completed the rst HAD questionnaire, 34 (67%) completed the second,
and 34 (67%) completed the third. Of 37 patients, 23 (62%) improved
in functional improvement over the study period, and 5 patients (13%)
deteriorated. Six of 11 of the patients (55%) who functionally deteriorated
through the study did not complete the HAD scale. Five of 38 patients
(13%) were anxious postsurgery, and four of these patients continued to be
anxious throughout the study period. Eight of 34 (23%) were anxious pre-
radiotherapy. All patients reporting heightened levels of anxiety were aged
65, and the anxiety levels were not related to initial functional impairment
or change in function. Five patients had a signicant depression at one or
more time between surgery and radiotherapy. Four of the 5 patients with
depression had a past history of depression. Anxiety was more common in
younger patients. Anxiety was slightly more frequent pre-radiotherapy. A
past medical history of depression is a predictor of signicant depression in
the postoperative period.
124. ADVERSE EFFECTS OF ANTIEPILEPTIC DRUGS IN
BRAIN TUMORS: PRELIMINARY DATA OF A MULTI-
INSTITUTIONAL SURVEY. ARE NEW ANTIEPILEPTIC
DRUGS LESS TOXIC?
M. Maschio, M. Riva, R. Sofetti, R. Rud, M. Scerrati, A. Mauro,
C. Bompressi, L. Provinciali, F. Imbesi, C. Carapella, A. Pace, and B.
Jandolo, on behalf of the Italian Association of Neuro-Oncology (AINO)
and the Italian Society of Neurology (SIN)
Adverse effects of antiepileptic drugs (AEDs) in patients affected by
brain tumors are more frequent than in nontumoral epileptic patients (24%
vs. 12%, respectively, Glantz et al). Several classical AEDs (phenobarbital,
phenytoine, and carbamazepine) are potent enzyme-inducing drugs, have
serious CNS side effects that may alter quality of life and interfere with anti-
cancer treatments, and may presents hematological and systemic toxicities.
There are few studies focused on incidence of side effects from new AEDs
in neuro-oncology. We report preliminary data of an Italian multicentric
study based on antiepileptic treatment of a large population of brain tumor
patients treated with new AEDs. The aim of the study was to evaluate the
incidence of adverse effects in brain tumor patients treated with oxcar-
bazepine (OXC), lamotrigine (LTG), vigabatrin (GVG), topiramate (TPM),
gabapentin (GBP), and levetiracetam (LEV). Two hundred thirty patients
were analyzed (148 M, 93 F), mean age 50 years (range, 1680). Histol-
ogy was glioblastoma in 112 patients, anaplastic astrocytoma in 59, and
low-grade glioma in 68. Patients were treated in monotherapy in 170 cases:
OXC, 69; LTG, 40; GVG, 9; TPM, 51; and GBP, 1. In 60 cases, patients
were treated in polytherapy with new AEDs in add-on to classical AEDs (PB
in 26 patients; CBZ, 26; OXC, 75; TPM, 76; LTG, 52; GVG, 11; DHT, 11;
LEV, 10; VPA, 3; GBP, 1). Overall incidence of adverse effects was 13.5%
(31/230 patients). The incidence of side effects in patients treated in mono-
therapy with new AEDs was 7.0% (12/170). More frequent side effects
included skin reaction in 17 (7.4%), mostly in patients treated in associa-
tion with carbamazepine or phenobarbital; other side effects were sedation,
weight loss, hyponatriemia, and liver toxicity. In 14 patients, the severity
of side effects led to discontinuation of treatment. Despite the limits of a
retrospective analysis from a multi-institutional series of patients, our data
seems indicate that new AEDs are better tolerated than standard antiepi-
leptic drugs. Given the relevant inuences of anticonvulsant treatment on
quality of life of brain tumor patients, and the important interactions with
anticancer treatments, larger studies are needed to compare tolerability and
efcacy of new AEDs with respect to standard AEDs.
125. PHASE 2 STUDY OF TOPOTECAN IN COMBINATION
WITH CONCURRENT RADIOTHERAPY IN ADULTS WITH
GLIOBLASTOMA WITH INCOMPLETE RESECTION
T. Lesimple,
1
M. Ben Hassel,
1
D. Gdouin,
1
J. Bay,
2
D. Frappaz,
3

C. Linassier,
4
G. Piot,
5
M. Fabbro,
6
S. Saikali,
7
and Y. Gugan
7
;
1
Neurooncology, Rennes;
2
Medical Oncology, Clermont-Ferrand;
3
Pediatric Oncology, Lyon;
4
Medical Oncology, Tours;
5
Medical
Oncology, Le Havre;
6
Medical Oncology, Montpellier;
7
Neurosurgery,
Rennes; France
In a previous phase 1 study of topotecan (TPT) administered as
a continuous infusion (CIV) for 5 days every 2 weeks in combina-
tion with concurrent radiotherapy (RT), the recommended dose of
TPT was 0.9 mg/m
2
/day (Lesimple et al., J. Neurooncol. 65, 2003).
The antitumor activity (measured by 12-month overall survival [OS]) and
the safety of TPT were assessed in patients (pts) with histologically proven
and previously untreated glioblastoma multiforme (GBM): After partial
resection or stereotactic biopsy, pts received cranial RT (60 Gy/30 frac-
tions/40 days) and 3 cycles of 0.9 mg/m
2
/day of TPT (Hycamtin, Glaxo-
SmithKline, Marly le Roi, France) as CIV from day 1 to 5 on weeks 1, 3,
and 5 during RT. A total of 50 pts were entered, and 37 pts (M/F: 24/13;
median age: 59, range: 42-69; PS 0/1-2: 17/20) were analyzed here. Twenty-
one pts had stereotactic biopsy and 16 had partial resection. Grade 34
hematological toxicity was observed in 15 patients: neutropenia (16%,
associated with fever in 4%), lymphopenia (15%), thrombopenia (5%) and
anemia (3%). Grade 34 nonhematological toxicity consisted of hypergly-
cemia (4 pts); vomiting, diarrhea, hypokalemia (2 pts); and hyponatremia,
GGT elevation, catheter-related infection, nausea, somnolence and throm-
botic microangiopathy (1 patient each). One patient experienced a partial
response (3.7% of 27 evaluable pts for response) and 11 pts (40.7%) had
stabilization, with a time to progression of 12 weeks. One-year OS rate
was 41.7% for the 37 pts (median OS, 41 weeks). TPT in combination with
RT was well tolerated but had modest activity in partially resected GBM in
terms of response rate and 12-month OS.
126. TREATMENT WITH 3-WEEK COURSES OF DAILY
TEMOZOLOMIDE AS SECOND-LINE CHEMOTHERAPY OF
RECURRENT HIGH-GRADE GLIOMAS
R. Gaspar, A. Guerrero, and J. Ponce; Department of Medical Oncology,
Hospital Universitario La Fe, Valencia, Spain
Several studies suggest that continuous treatment with temozolomide
(TMZ) may be more effective than the standard 5-day schedule. We pre sent
our results with 3-week courses, daily TMZ, in recurrent high-grade glio-
mas (HGG). Between March 2003 and September 2004, 17 patients (pts)
were included. Patient characteristics were as follows: 9 males, 8 females;
median age, 39 years (range, 1859); histology: 7 glioblastomas, 4 ana-
plastic astrocytomas, 1 anaplastic ependymoma, and 5 suspected HGG
by neuro-imaging and clinical criteria. Previous treatments of recurrence
were as follows: TMZ (200 mg/m
2
for 5 d every 28 d) in 13 cases; CPT-11
(125 mg/m
2
d, 1, 8, 22, and 29 every 36 d) in 3 cases; PCV in 1 case. At
progression, pts were treated with TMZ, 75 to 80 mg/m
2
/d for 21 d every 4
weeks. Ninety cycles of TMZ were administered (median, 4 cycles; range,
211 cycles). No grade 3 or 4 toxicity has been observed. According to
Macdonalds criteria, partial response was achieved in 2 pts, stable disease
was achieved in 9 pts, and 5 pts progressed. Both partial responses, lasting
4 and 6 months, have been observed in patients who progressed during
treatment with standard 5-day courses of TMZ. Nine of the 17 pts are
still on treatment; 4 additional pts have been recently included. We con-
clude that TMZ given continuously for 21 d every 4 weeks is well tolerated.
Responses can be achieved in pts who have progressed with the standard
5-day schedule.
127. PREDICTORS OF SURVIVAL AND TUMOR
RECURRENCE IN A COHORT OF 530 PATIENTS WITH
GLIOBLASTOMA MULTIFORME: A REPORT FROM
THE BRAIN CANCER REGISTRY OF THE NATIONAL
NEUROLOGICAL INSTITUTE CARLO BESTA, ITALY, 1997
TO 2002
G. Filippini, M. Farinotti, C. Falcone, C. Politi, and R. Farina; Istituto
Nazionale Neurologico, Neuroepidemiology, Milan, Italy
Glioblastoma multiforme (GBM) is the most frequent brain tumor and
accounts for approximately 12%15% of all intracranial neoplasms and
50%60% of all glial tumors. Aims of our study were to examine if resec-
tion is better than biopsy, to verify the role of extent of resection in relation
to survival and tumor recurrence/progression in patients with GBM, and
to evaluate the added benecial value of radiotherapy and chemotherapy.
All patients were consecutively registered from January 1997 to December
2002 in the Brain Cancer Registry of the National Neurological Institute
Carlo Besta, Milan. During this period, all adult patients newly diagnosed
with histologically veried cerebral GBM were included and followed up
until December 2003. Data about Karnofsky performance status (KPS),
type of initial surgical procedure, therapies, survival, and recurrence of
disease were studied. Analysis was done using both Cox and logistic regres-
sion models. Five hundred thirty patients (60% males, 40% females) were
included; mean age at intervention was 56 years (1681 years). Median clin-
ical follow-up time was 48 weeks. Median survival time was 58 wks (95%
CI, 5462). Survival probability was 55% at 1 year and 7% at 3 years. In
multivariate analysis, age resulted as one of the most signicant prognostic
factors for survival of pts with GBM, with a hazard ratio (HR) of 1.32
(95% CI, 1.041.68) for age 53 to 61 years and 1.83 (95% CI, 1.402.39)
for age 61 years compared with 15 to 52 years (reference category). The
intervention most signicantly associated with survival was radiotherapy
with HR 4.17 (95% CI, 2.985.85) for no radiotherapy. With respect to
extent of resection, HR was 2.05 (95% CI, 1.363.09) for biopsy and 1.25
(95% CI, 0.901.74) for partial resection (reference category: total resec-
tion). Median time to recurrence/progression (TTP) was 24 weeks (95% CI,
2226). In multivariate analysis, the most signicant prognostic factor for
TTP was radiotherapy with HR 3.86 (95% CI, 2.855.22) for no radio-
therapy. Biopsy, compared to surgical intervention, was signicantly associ-
ated with the following: sex (females vs. males), with a relative risk (RR)
of 2.15 (95% CI, 1.163.98); number of lesions (multiple vs. single), with
RR 4.04 (95% CI, 2.038.05); tumor size (overlapping lesion vs. single
lesion), with RR 3.66 (95% CI, 1.887.12); and side (bilateral/median
vs. unilateral), with RR 7.92 (95% CI, 3.3618.65). Partial resection,
compared to a total one, was signicantly associated with KPS (70 vs.
70), with RR 2.34 (95% CI, 1.154.79), and tumor size (overlapping
lesion vs. single lesion), with RR 1.97 (95% CI, 0.983.96). We analyzed
data from the largest data set on GBL in Italy. Radiotherapy and total surgi-
cal resection were signicant independent predictors of survival of patients
with GBM, but measurements of morbidity and quality of life associated
with treatments are now critical issues for these patients.
128. EPILEPTIC SEIZURES DURING FOLLOW-UP OF
PATIENTS TREATED FOR PRIMARY BRAIN GLIOMA
J. Hildebrand, C. Lecaille, J. Perennes, and J. Delattre; Hpital de la
Salptrire, Paris, France
Epileptic seizures are common in patients with brain glioma and may
signicantly alter the quality of life of these patients. The aim of our study
is to determine the presentation, the incidence, and the severity of epileptic
seizures in follow-up of patients treated for primary glioma. Two hundred
thirty-four consecutive patients attending an outpatient clinic for chemo-
therapy of supratentorial brain glioma were examined. One hundred eighty-
three (78.2%) experienced tumor-related seizures, and 51 (22.8%) did not.
All epileptic patients were on antiepileptic drugs (AEDs). Compared with
patients without epilepsy, the group of epileptic patients was characterized
by a higher proportion of low-grade gliomas (epilepsy in the course of the
malignant disease. Generalization occurred in 50% of early seizures, but in
only 19.1% of patients with seizures persisting after the initiation of AEDs
and specic antitumor therapies. The reduction of seizure generalization
was statistically signicant (P 0.001). Despite AED administration and
various antitumoral treatments, half of the patients had seizure within one
month and two thirds within three months preceding the last evaluation.
Most tumor-related seizures rst appear in the early course of the disease,
usually as a presenting manifestation. AEDs combined with specic anti-
tumoral treatments signicantly reduce the rate of seizure generalization.
However, the majority of patients continue to present mostly focal epilepsy
during therapeutic follow-up.
129. IMAGING RESPONSE IN PHASE 2 TEMOZOLOMIDE
TRIALSREPORT INITIAL TUMOR SIZE
C.F. Schwindack,
1
C. Graham,
1
and R. Grant
2
;
1
Wellcome Trust Clinical
Research Facility;
2
Division Clinical Neurosciences, Western General
Hospital, Edinburgh, Scotland, UK
Imaging response is an important measure in most trials of malignant
glioma. In a previous paper of RMP-7 and carboplatinum, we found age
was related to tumor grade and initial size of tumor in glioblastoma (GBM),
but not anaplastic astrocytoma (AA). Initial tumor size was important for
response in GBM, and age was linked to speed of response in AAs, but
numbers were too small in the GBM group to allow analysis. This study
analyzes imaging response data from three trials of temozolomide. Imag-
ing response data from 3 clinical trials of temozolomide were supplied by
Schering Plough Research International (SPRI).

Inclusion criteria included
recurrent supratentorial AA or GBM, age 18 years, and Karnofsky per-
formance status 70. Temozolomide was administered during the rst 5
days of a 28-day cycle (200 mg/m
2
/day or 150 mg/m
2
/day if prior chemo-
therapy). Macdonald imaging criteria for response were used. Speed of
response (fast/slow) was determined by tumor size after 2 cycles. Change
in size and speed of response to treatment were related to size, age, initial
tumor size, and tumor type. Data on 301 patients were analyzed. There
were 108 patients with AA (median age, 41.5 years) and 193 GBM (median
age, 55 years) with a signicant age difference (P 0.001; 95% CI, -15.0 to
-9.00). Median Karnofsky was 85 for AA and 80 for GBM (P 0.03; 95%
CI, 0.0010.00). Median initial tumor size was 9.94 cm
2
in AA and 15.51
cm
2
in GBM (P 0.001; 95% CI, -6.40 to -1.65). Of the patients with AA,
34% showed a response compared to 9.7% with GBM (odds ratio, 0.20;
95% CI, 0.100.40; P 0.0001). Size: Initial median tumor size was 8.48
cm
2
in responders with AA versus 10.81 cm
2
in nonresponders (P 0.59;
95% CI, -5.102.50)

and 6.25 cm
2
in responders with GBM versus 15.63
cm
2
in nonresponders (P 0.04; 95% CI, -10.75 to -0.32). There was no
statistically signicant difference in median initial tumor size of those with
a fast response compared with a slow response (AA, P 0.22; GBM, P
0.83). Age: There was no association between age and initial tumor size in
AAs (P 0.40) or GBMs (P 0.96). GBM patients who were responders
were younger (P 0.01; 95% CI (214), but median ages for responders
with AA were no different than nonresponders (P 0.57; 95% CI, -7.4).
Age did not inuence speed of response: AA (P 0.20; 95% CI, -124)
or GBM (P 0.94; 95% CI, -1818). Patients with a GBM are likely to be
older and have larger tumors. Patients with a small GBM are more likely to
respond than those with a large GBM. Within the tumor group, however,
there was no relationship between age and initial tumor size. Studies evalu-
ating response should record age and initial tumor size, and trials should
stratify for it.
130. PRIMARY TEMOZOLOMIDE CHEMOTHERAPY IN
ELDERLY PATIENTS WITH PRIMARY CNS LYMPHOMA
(PCNSL)
U. Herrlinger, M. Uhl, and M. Weller; University of Tbingen, General
Neurology, Tbingen, Germany
In some PCNSL patients over 60 years, standard high-dose methotrex-
ate (HD-MTX) therapy may not be applicable due to comorbidities such
as impaired renal function. This prompts the search for other chemothera-
peutic agents that can be applied to these patients instead of HD-MTX.
Progression-free survival, overall survival, and toxicity were retrospectively
analyzed in patients with primary CNS lymphoma treated with temozolo-
mide alone as primary therapy. We report 7 patients who had histologically
conrmed PCNSL (n 6) or a steroid-responsive lesion highly suggestive
of PCNSL by neuroimaging (n 1). The patients (6284 years old) received
1 to 8 four-week courses of temozolomide (200 mg/m
2
for 5 days in 6
patients, reduced dose of 150 mg/m
2
in 1 patient). The median number
of courses applied was 3. Complete responses (CRs) were achieved in 4
patients, 1 patient had stable disease after 3 courses and therapy was then
switched to WBRT due to myelosuppression, 2 patients had primary pro-
gressive disease and did not receive any further therapy. In the patients
achieving CR, CR persisted for 5, 18, 21 and 48 months. After a median
follow-up time of 17 months (range, 148 months), median survival has
not been reached yet. One patient died after 1 month due to tumor pro-
gression, and 6 patients are alive. Acute toxicity consisted of high-grade
thrombopenia and leukopenia with subsequent infection in one patient who
had received 200 mg/m
2
temozolomide and high-grade leukopenia without
further complications in another patient. Temozolomide appears to be an
effective and tolerable therapy for elderly patients with PCNSL and comor-
bidity who cannot receive HD-MTX.
131. ADJUVANT CHEMOTHERAPY WITH TEMOZOLOMIDE
AND LIPOSOMAL DOXORUBICINE IN THE FIRST-LINE
THERAPY OF PATIENTS WITH GLIOBLASTOMA: A PHASE
2 TRIAL
P. Hau, T. Jauch, U. Baumgart, D. Beier, S. Gnbauer, H. Koch, C.
Wismeth, A. Steinbrecher, and U. Bogdahn; Department of Neurology,
University of Regensburg, Regensburg, Germany
Temozolomide (TMZ; Temodal/Temodar) recently showed promising
results in the rst-line therapy of glioblastoma (Stupp, 2004). Pegylated
liposomal doxorubicin (PEG-Dox; Caelyx/Doxil) was successfully evalu-
ated in patients with recurrent high-grade glioma (Fabel, 2001; Hau, 2002).
Therefore, a combination of both agents seems promising. Here, we update
data on a pilot phase 2 trial using this regimen. We initiated a combina-
tion regimen consisting of TMZ and PEG-Dox in the rst-line therapy of
patients with glioblastoma. TMZ is given during standard radiotherapy
(initiation) and on days 1 to 5 in 28 days starting 4 weeks after completion
of radiotherapy (maintenance). PEG-Dox is given as a short-time infusion
in a dose-escalation regimen once prior to radiotherapy (initiation) and on
days 1 and 15 starting 4 weeks after completion of radiotherapy (mainte-
nance). PEG-Dox is escalated for 5 mg/m
2
in groups of three patients with
a highest dose of 20 mg/m
2
(group 4). Primary end point is dose-limiting
toxicity (DLT) in the toxicity phase and time to progression in the efcacy
phase. In the rst treatment group (5 mg/m
2
of PEG-Dox), one out of 7
evaluable patients had a dose-limiting toxicity (DLT). In the second, third,
and fourth treatment groups using 10, 15, and 20 mg/m
2
of PEG-Dox, the
regimen was tolerated without DLT. Concerning efcacy in the treated-
patients analysis of 17 patients treated so far, 1 had a partial response in
MRI, and 12 patients had tumor stabilization 4 weeks after conclusion of
radiotherapy. As no DLT was observed in dose group 4, the efcacy phase
of the study will be performed with 20 mg/m
2
of PEG-Dox. Accrual started
in November 2004. Considering the results of the dose escalation phase of
this study, the regimen is feasible, tolerable, and able to induce objective
responses and stabilizations in patients with glioblastoma using the stan-
dard dose of TMZ and 5 to 20 mg/m
2
of PEG-Dox.
132. SEVERE MYELOSUPPRESSION WITH THE FIRST
COURSE OF STANDARD DOSE TEMOZOLOMIDE:
THE X FACTOR
T.S. Armstrong, R. Manning, and M.R. Gilbert; Neuro-Oncology, The
University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA
Although myelosuppression is a dose-limiting toxicity of most cyto-
toxic chemotherapies, a reported benet of temozolomide is that myelo-
suppression is relatively uncommon. Most studies of temozolomide report
an overall incidence of grade 34 myelosuppression of 5% to 8%. In the
initial trial of 445 cancer patients treated with temozolomide, an 11%
incidence of signicant myelosuppression was reported in females, with
older female patients who received higher doses having a greater chance
of developing both neutropenia and thrombocytopenia. In our practice,
we recognized a trend toward female brain tumor patients having a higher
incidence of myelosuppression during the rst course of chemotherapy with
conventional dose temozolomide. Therefore, we analyzed the hematologic
toxicity in 18 consecutive patients after their rst course of treatment with
standard dose temozolomide (200 mg/m
2
, days 15 of a 28-day cycle). A
marked difference in the incidence of clinically signicant myelosuppression
(grade 3 or 4) was noted between male 14% (1/7) and female 46% (5/11)
patients. The solitary male patient with myelosuppression experienced both
a grade 4 thrombocytopenia and neutropenia. Of the ve female patients
with myelosuppression, 4/5 experienced a grade 4 neutropenia and 1/5 a
grade 3. Two out of ve experienced a grade 4 thrombocytopenia and 2/5 a
grade 3. Furthermore, four females required hospitalization for febrile neu-
tropenia, whereas none of the male patients developed this complication.
Among Caucasian females, 71% (5/7) developed severe myelosuppression,
compared with none of the four nonwhite females. This higher incidence of
toxicity was not associated with a higher body surface area (BSA), serum
creatinine, serum chemistry, smoking status, or concomitant medications.
These intriguing preliminary ndings prompted a thorough review of the
MDACC brain tumor treatment database that is currently under way and
will be reported.
133. NATURAL HISTORY AND PROGNOSTIC FACTORS OF
GANGLIOGLIOMAS
A. Carpentier and A. Behin; Hpital de la Salptrire, Fdration
de Neurologie Mazarin, Paris; Ganglioglioma Study Group of the
Association des Neuro-Oncologues dExpression Franaise; France
Gangliogliomas are rare neuroepithelial tumors. This study led by the
ANOCEF (a French-speaking association of neuro-oncologists) aims at a
better assessment of the features and prognostic factors of gangliogliomas.
This retrospective study reviewed 176 cases from 9 French teaching hospi-
tals, diagnosis being based on neuropathological records. A central review
of cases was proposed. Signicant data, including symptoms, pre- and
post-operative neuroimaging, pre- and post-operative evolution, surgery
and other treatments, were all recorded. Potential prognostic factors for
progression were tested in univariate analysis using log-rank tests and in
multivariate analysis using a Cox model. Results concern the rst 120 cases
of the study; nal presentation will include updated gures on the whole
series. Median age at onset was 17 years (range, 40 days68 years). Epilepsy
was the revealing symptom in 75% of cases. On CT scan and MRI, 65% of
tumors were partly cystic, with contrast enhancement in more than 90% of
cases. Edema was absent or not signicant in 82% of cases; 92% of cases
were supratentorial, mostly temporal. Complete resection was performed
in 65%, partial resection in 24% of patients. After surgery, epilepsy disap-
peared or was signicantly reduced in 45% and 33% of cases, respectively.
Only 4% of tissue samples showed anaplastic features. Recurrence or pro-
gression was observed in 19% of patients. Surgery was performed in 74%
of those cases. Nonsurgical therapy included radiotherapy (17 patients) and
chemotherapy (7 patients). Initial prognostic factors for recurrence/pro-
gression in univariate analysis included incomplete resection (P 0.001),
anaplastic features (P 0.0001), presence of edema on imaging (P 0.03)
and nonepileptic onset (P 0.0001), the latter remaining the only signi-
cant prognostic factor in multivariate analysis. Age, sex, tumor size, cystic
or calcied components did not inuence recurrence or progression. On
the whole, 63% of patients were progression-free 5 years after diagnosis;
median survival was not reached. Most gangliogliomas share common,
homogeneous features. The frequency of contrast enhancement on diagno-
sis should be underlined. The importance of initial resection is conspicuous.
On recurrence, surgery remains the standard option. Data pertaining to
radiotherapy and chemotherapy are too sparse to allow precise recommen-
dations and warrant further studies.
134. DELAYS IN RADIATION THERAPY AFTER SURGERY
FOR INTRINSIC BRAIN TUMOR ARE ASSOCIATED WITH
CLINICAL DETERIORATION IN ELDERLY PATIENTS
L. Kilbride,
1
I. Whittle,
2
and R. Grant
1
;
1
School of Acute & Continuing
Care Nursing, Napier University, Edinburgh;
2
Centre for Neuro
Oncology, Lothian Universities NHS Trust, Edinburgh; Scotland
Benecial effects of steroids and surgery are often counterbalanced by
brain tumor progression in the interval between surgery and radiotherapy.
We prospectively studied a cohort of patients examined post surgery and
pre-radiotherapy to study the inuence of the following: age (60 vs. 60
years), dexamethasone dose (mg), and time interval until start of radiation
(5 weeks vs. 30 weeks). Fifty-one consecutive patients with an intrinsic
brain tumor consented to be prospectively, objectively assessed postsurgery
and immediately preradiotherapy using the Edinburgh Functional Impair-
ment Test (EFIT). The EFIT consists of the nine-hole peg test (NHPT),
timed 10-m walk (TMW), Williams Delayed Recall Test (WDRT), and
Boston Aphasia Severity Rating Scale (BASRS). Normal values and val-
ues for clinically signicant change for these tests have previously been
published. Patients were grouped as (a) no decits, (b) limb decits only,
(c) memory decits only, (d) combination of limb and memory/speech. An
initial dose of dexamethasone 12mg/day was arbitrarily considered high
dose. A waiting time of 5 weeks was considered to be delayed. Of 51
patients, 49 (96%) completed EFIT at both time intervals. There were 28
males and 13 females, 34 patients were 60. Median age was 55; 81% of
patients had high-grade glioma, 19% other. Also, 96% were treated with
steroids (39% high dose); 45% of patients had an abnormal NHPT, 76%
had abnormal TMW, 43% had abnormal WDRT, and 41% had abnormal
BASRS after surgery. Younger patients (91%) as opposed to older patients
(81%) were more likely to have a degree of functional impairment postsur-
gery; 51% of patients improved and 22% deteriorated prior to radiotherapy.
Patients 60 years were more likely to improve prior to radiotherapy (59%
vs. 33%). Older patients were more likely to deteriorate (40% vs. 15%).
There was no clear association between initial or mean daily dose of dexa-
methasone and type or degree on neurological impairment, although fewer
patients who deteriorated with memory or speech impairment were given
high doses of dexamethasone than those with limb impairment. Only 37%
started radiation therapy within 5 weeks. No patient treated within 5
weeks deteriorated compared with 36% 5 weeks. We conclude that most
young patients with functional impairment post-surgery for intrinsic brain
tumors improve in the period up to radiation therapy. Older patients and
patients waiting more than 5 weeks are more likely to have clinical deterio-
ration. Initial high-dose dexamethasone seems to have no clear advantage
over lower doses in the postoperative period. Delays in radiotherapy are
associated with clinical (and probable radiological) tumor progression
135. FIRST-LINE CHEMOTHERAPY WITH TEMOZOLOMIDE
IN RECURRENT/PROGRESSIVE OLIGODENDROGLIAL
TUMORS: A PHASE 2 STUDY
R. Rud, A. Costanza, E. Laguzzi, E. Trevisan, M. Nobile, and R.
Sofetti; Neuro-Oncology Service, University of Torino, Neuroscience,
Torino, Italy
There are few data regarding the efcacy of rst-line temozolomide in
recurrent oligodendroglial tumors, and the responses range from 17% to
54%. The purpose of this phase 2 study was to investigate the efcacy and
toxicity of temozolomide standard schedule in patients with oligodendro-
glial tumors at rst relapse after surgery alone or surgery and radiotherapy.
The inclusion criteria were as follows: age 18 yrs, Karnofsky performance
status 60; histological diagnosis of oligodendroglioma or oligoastrocy-
toma (grade II or III according to WHO classication); tumor progression
on MRI; chemotherapy nave. Temozolomide was administered at 200 mg/
m
2
for 5 days in cycles of 28 days up to a maximum of 24 cycles in respond-
ing or stable patients or to unacceptable toxicity. Primary end point of the
study was response basing on Macdonalds criteria. Thirty-four patients
are assessable, 24 males and 10 females, with a median age of 47 (range,
1873). A median of 10 cycles (range, 124) were administered. Responses
were as follows: CR, 2/34 (6%); PR, 10/34 (29%); SD, 21/34 (62%); PD,
1/34 (3%). Among patients with SD, 3 had a reduction of tumor volume of
20% to 40% (minor response). The overall response rate (CR PR) was
35%. The maximum tumor response was observed after 3 cycles in 2/12, 6
cycles in 7/12, 9 cycles in 2/12, and 15 cycles in 1/12. Four of 12 responding
(CR PR) patients (33%) and 5 of stable patients (24%) displayed a sig-
nicant reduction of seizures. Nine of 23 (39%) pure oligodendrogliomas
responded, including the 2 patients with CR (grade III tumors), compared
to 3/11 (27%) oligoastrocytomas. Ten of 22 enhancing tumors responded
(45%) compared to 2/12 nonenhancing tumors (17%), and 47% of grade
III responded compared to 26% of grade II. Median TTP was 14 months
(range, 229), with a PFS at 6 months of 88% and at 12 months of 59%.
Grade IIIIV myelotoxicity was observed in 26% of patients. Temozolo-
mide shows activity as rst-line treatment in oligodendroglial tumors at
rst relapse (CR PR minor response: 44%). Pure oligodendrogliomas
and enhancing and high-grade tumors tend to respond better. A long-term
treatment with temozolomide is well tolerated.
136. PHASE 1 CLINICAL TRIAL AND PHARMACOKINETIC
STUDY OF KARENITECIN IN THE TREATMENT OF
RECURRENT MALIGNANT GLIOMAS: A STUDY OF THE
NABTT CNS CONSORTIUM
S. Grossman, J. Supko, K. Carson, S. Phuphanich, T. Batchelor, D.
Peereboom, B. Nabors, G. Lesser, S. Desideri, and F. Hausheer. NABTT
CNS Consortium, Baltimore, Maryland, USA
Camptothecins have clinical activity in colorectal, ovarian, lung and,
to a lesser extent, brain cancers, but their use has been limited by gas-
trointestinal and hematopoetic side effects. Karenitecin is a highly lipo-
philic camptothecin derivative, with a 2-(trimethylsilyl)ethyl substituent
at the 7-position, rendering the terminal lactone ring much more resistant
to inactivating hydrolysis under physiological conditions than most other
campotothecins. The pharmacokinetics of several camptothecin analogues
is dramatically affected by the concomitant use of hepatic enzyme-inducing
antiepileptic drugs (EIAEDs). This study was conducted to (1) determine
the maximum tolerated dose (MTD) of karenitecin in adults with recur-
rent malignant glioma, (2) describe the effects of EIAEDs on its pharmaco-
kinetics, and (3) obtain preliminary evidence of activity. Karenitecin was
administered intravenously over 60 min once a day for 5 consecutive days,
every 3 weeks. The starting dose was 1.00 mg/m
2
per day. The dose was
escalated by using the continual reassessment method independently in
cohorts stratied by EIAED use. Three patients were treated at each dose
level. Intrapatient dose escalation was not permitted. Treatment was con-
tinued until disease progression, treatment-related dose-limiting toxicity, or
patient withdrawal. Pharmacokinetic samples were obtained to dene the
total karenitecin plasma prole for the rst dose of drug. We accrued 32
pts (20 males) to this study. Their median age was 52 years (range, 3472),
median KPS was 90 (range, 60100); 78% of the patients had glioblas-
toma multiforme and the remainder had anaplastic gliomas. One prior che-
motherapy regimen was permitted. Dose levels evaluated in the EIAED
arm were 1.0, 1.5, 1.7, 1.9, and 2.1 mg/m
2
and in the EIAED cohort 1.0,
1.5, and 1.8 mg/m
2
. Myelosuppression was the major toxicity observed
(WBC platelets RBC), with a short-lived grade 3-4 neutropenia or
thrombocytopenia occurring in 28% and 16% of patients, respectively. The
MTD was determined to be 2.0 mg/m
2
in EIAED patients and 1.5 mg/m
2

in the EIAED patients, a difference of 25%. Statistical comparisons to
assess the inuence of EIAEDs were based upon data for patients treated
with 1.5 mg/m
2
where there were maximal patient numbers (7 EIAED,
6 EIAED). In comparison to the EIAED cohort, the mean (SD) maxi-
mum concentration of drug in plasma was 36% lower (22.9 12.1 vs.
35.8 ng/ml), and the total mean total body clearance was 29% higher
(13.5 13.5 vs. 10.5 3.3 l/h/m
2
) in the EIAED group. The median
survival after starting therapy was 6.5 months (95% CI, 4.09.7 months),
and 25 of the 32 patients are deceased. No complete or partial responses
were observed, although 12 patients were treated at or above the MTD.
As observed with other camptothecin analogues, karenitecin elimination
is enhanced by EIAED administration. Single agent karenitecin does not
appear very active in recurrent high-grade gliomas.
137. LONG-TERM TREATMENT WITH TEMOZOLOMIDE
IN HIGH-GRADE GLIOMAS IS FEASIBLE AND LEADS TO
LONG-TERM SURVIVAL IN A SIGNIFICANT SUBSET OF
PATIENTS
D. Beier,
1
P. Hau,
1
T. Jauch,
1
D. Koch,
2
T. Hundsberger,
3
S. Schuth,
4

and U. Bogdahn
1
;
1
Neurology, University of Regensburg, Regensburg;
2
Neurosurgery and
3
Neurology, University of Mainz, Mainz;
4
Essex
Pharma GmbH, Muenchen; and the Long-Term Temozolomide Study
Group; Germany
Although survival in patients with high-grade gliomas is relatively poor,
warranting intensied treatment approaches, postoperative radiochemo-
therapy is frequently limited by bone marrow toxicity, usually terminat-
ing chemotherapy after 6 cycles of chemotherapy. Clinical studies using
temozolomide (Temodar; TMZ) have produced promising results concern-
ing survival and long-term feasibility, which is in part due to the lacking
cumulative toxicity of TMZ. Nevertheless, no systematic data concerning
long-term feasibility of TMZ have been acquired yet. Within a retrospec-
tive eld analysis, German neuro-oncologists were approached to report
on their treatment strategies with TMZ in high-grade gliomas using a
standardized questionnaire. Long-term application of TMZ (more than 12
cycles or more than 12 months of any dose) were analyzed and evaluated
for feasibility, efcacy, and tolerability. Altogether, 128 patients with WHO
Grade III or IV gliomas who fullled the study criteria were identied by
49 neuro-oncologists. Most of the patients were treated with the standard
scheme using 150 to 200 mg/m
2
, and some were treated according to pro-
tocol EORTC 26981/22981 or by modied schemes. In rst-line treatment
(n 64), the median number of applied cycles was 13 (range, 1240),
with a mean progression-free duration of 56 weeks (range, 52160 weeks).
Recurrent patients (n 55) received a median number of 14 cycles (range 12
to 44 cycles), with a median duration of 62 weeks (range, 52176 weeks).
Grade 3 or 4 (NCI-CTC) toxicity concerning the gastrointestinal system
(n 7), leukopenia (n 13), thrombocytopenia (n 7), or infection (n
5) was observed only in a small amount of patients. Nevertheless, toxicity
data may underestimate the true incidence of toxicity due to the retrospec-
tive character of the study. Although this was a retrospective analysis, we
could identify a signicant number of patients with long-term application
of TMZ. Up to 44 cycles were given, and times of up to 160 weeks in the
rst-line and 176 weeks in the recurrent setting without signs of tumor pro-
gression could be reached, indicating that patients with active high-grade
gliomas may have well-controlled disease over signicant periods of time.
138. SUBCORTICAL MAPPING OF MOTOR TRACT
PATHWAYS IMPROVES SURGICAL REMOVAL OF HUMAN
GLIOMAS
L. Bello, F. Acerbi, C. Giussani, G. Carrabba, M. Fava, V. Songa,
P. Baratta, R.M. Villani, and S.M. Gaini; Neurosurgery, University of
Milano, Milano, Italy
Surgery for lesions involving motor areas or pathways requires the iden-
tication of functional cortical areas to reduce tumor resection morbid-
ity. Intraoperative identication of functional descending motor pathways
has been recently advocated as a promising technique to further reduce
postoperative morbidity. We present our experience with subcortical iden-
tication of motor functional tracts during awake surgery for removal of
gliomas involving motor tract pathways. There were 45 patients (25 males,
20 females, age ranging from 22 to 45 years) harboring a low-grade (39) or
high-grade (6) glioma located in the frontal lobe and involving motor cortex
and/or pathways. In 23 patients, the tumor was also involving language
areas and/or pathways. Cortical and subcortical mapping was performed by
the use of an Ojemann stimulator and using the largest current that did not
produce afterdischarge. Cortical areas were found in all cases. Subcortical
stimulation by using the same current threshold was also applied during
tumor resection. Subcortical stimulation mapping was alternated by tumor
resection in a back and forth fashion. Motor response was monitored clini-
cally as the appearance of an involuntary motor response or by the use of
a continuous multichannel EMG recording. For lesions involving language
tracts, patients were submitting to periodic counting, confrontation nam-
ing, and verbal generation tests. Subcortical motor pathways were found
in 55% of patients, and this was associated with a transient neurological
decit in 62.5%. If subcortical sites were not found, the chance of tempo-
rary decits was 8%. Subcortical language sites were identied in 23.8% of
cases, with 1 permanent decit. In case of decit, the resection was stopped,
the patient asked to rest. When the tasks were normalized, subcortical stim-
ulation was reapplied. In case of response, resection continued in the neigh-
boring structures. Patient fatigue was observed in 20% of cases. Extent of
removal was total in 68% and subtotal in 15% of cases. Electrical seizures
occurred in 2 patients and were successfully controlled with cold water irri-
gation. Repeated subcortical stimulations should be alternated with tumor
resection to follow white bers closely. Identication of subcortical sites
is associated with a higher risk of transient postoperative decits. Postop-
erative decits are low. Repeated subcortical stimulation further improves
safety of resection of tumor involving motor pathways.
139. OUTCOME OF PATIENTS UNDERGOING SURGERY
FOR BRAIN METASTASES
F. Bokstein
1
and Z. Ram
2
;
1
Neuro-Oncology Service and
2
Neurosurgery,
Tel Aviv Sourasky Medical Center, Tel Aviv, Israel
Brain metastases (BM) represent one of the most severe complications
of solid cancers. Surgical treatment of this debilitating condition, if feasible,
seeks not only to prolong the survival but also to improve immediately
the neurological condition and quality of life of the patients. Our objec-
tive is to report the results of surgical treatment of 82 consecutive patients
with BM treated in Tel Aviv Sourasky Medical Center since March 2003.
Eighty-two patients (47 females and 35 males) with the median age of 61
(range, 4482) underwent surgical removal of BM. The most common pri-
mary cancers were NSCLC (33 pts, 40%) and breast cancer (13 pts, 16%).
In 20 patients (24%) BMs were rst presentation of their malignancy. At
diagnosis of BM, the majority of patients (58, 71%) had active systemic
disease. Nine patients (11%) already had BM in the past, and 7 of them
(9%) received previously neurosurgical treatment. Sixty patients (73%) had
single BM; in 7 patients (9%) there were 2 BM; 14 patients (17%) suffered
from multiple (2) BM. In most cases of multiple BM only one (rarely
two) symptomatic lesion(s) responsible for neurological deterioration were
removed. The operation resulted in immediate neurological improvement in
72 patients (88%). The condition of 7 patients (9%) did not change. Three
patients (3%) died within 2 weeks after the operation. Surgical removal of
BM succeeds to achieve in most cases an immediate clinical improvement
and should be considered in neurologically symptomatic patients, including
patients with active systemic disease and multiple brain metastases.
140. PATTERN OF RECURRENCE IN PATIENTS WITH
GLIOBLASTOMA MULTIFORME TREATED WITH AN
ANTIANGIOGENIC THERAPY
J. Tuettenberg,
1
R. Grobholz,
2
R. Erber,
1
T. Korn,
1
F. Wenz,
3
and P.
Vajkoczy
1
;
1
Neurosurgery;
2
Pathology;
3
Radio-Oncology, Klinikum
Mannheim der Universitat Heidelberg, Mannheim, Germany
Glioblastoma multiforme is the prototype of an angiogenic tumor and
thus predestined for an antiangiogenic therapy. But a possible escape mech-
anism of the tumor cells is increased invasion. The aim of this study was
to evaluate the progression free- and overall survival in patients with glio-
blastoma multiforme, treated with a continuous, low-dose chemotherapy
with temozolomide and rofecoxib, with special respect to the localization
of tumor recurrence. Twenty-two patients with glioblastoma multiforme
received after operation and radiation therapy a continuous, low-dose che-
motherapy with temozolomide (up to 20 mg/m
2
) and the COX-II inhibi-
tor rofecoxib. Clinical and MRI follow-up examination was done every 8
weeks. Mean follow up time was 20 months. Tumor tissue was analyzed
for microvessel density, COX-II expression, and VEGF expression in 13
patients. Mean progression-free survival of all patients was 9.7 months, and
mean overall survival was 16.9 months. Of 22 patients, 14 (67%) suffered a
tumor recurrence distant to the original tumor localization. These patients
had a slightly shorter overall survival. Patients with a higher microves-
sel density responded signicantly better to the therapy (PFS 11.7 vs. 6.7
months). There was no relationship of the immunohistochemical mark-
ers and the incidence of distant tumor recurrence. Despite the dramatic
increase in distant tumor recurrences compared to historical controls, the
continuous, low-dose chemotherapy seems to be a promising therapy option
in highly vascularized glioblastoma, since the progression free-survival and
the overall survival compare very well to actual studies.
141. PHASE II STUDY OF CLORETAZINE FOR THE
TREATMENT OF ADULTS WITH RECURRENT
MALIGNANT GLIOMA
A. Desjardins, J. Vredenburgh, M. Badruddoja, J. Quinn, D. Reardon,
J. Rich, S. Sathornsumetee, K. Penne, D. Bigner, and H. Friedman; Duke
Alkylating agents are an important class of chemotherapeutic
drugs used for the treatment of CNS neoplasms. A novel class, the 1,2-
bis(sulfonyl)hydrazines (BCHs), has been synthesized. The BCH com-
pounds produce chloroethylating species but do not generate vinylation
or hydroxyethylation events unlike the nitrosoureas. The chloroethylat-
ing species preferentially alkylates the O
6
-position of guanine. Cloreta-
zine is a novel BCH compound. In preclinical studies of xenograft mod-
els of malignant gliomas Cloretazine has demonstrated potent antitumor
effect. In the HT29 tumor cell line that expresses O
6
-alkylguanine alkyl-
transferase (AGT), high concentrations of Cloretazine had similar cyto-
toxicity compared to a cell line that does not express AGT. We performed
a phase 2 study of Cloretazine in adult patients with recurrent malignant
glioma. Eligibility included adult patients with recurrent malignant glioma.
Patients were divided into two strata: stratum 1, recurrent/progressive glio-
blastoma multiforme (GBM), and stratum 2, recurrent/progressive anaplas-
tic astrocytoma (AA) or anaplastic oligodendroglioma (AO). Each patient
was treated with a 15-min infusion of Cloretazine at 300 mg/m every six
weeks. Responses were assessed by functional and imaging criteria after
one cycle (6 weeks). Patients were treated until disease progression or unac-
ceptable toxicity. The primary end points were to determine the activity,
the toxicity, the time to progression, and survival of patients with recurrent
malignant glioma treated with Cloretazine. To date, 38 patients have been
enrolled, 32 in stratum 1 (planned accrual [PA] 32), and six in stratum 2
(PA 38), for a median age of 55.5 years (range, 3168) and a 71% male
population. Thirty-six patients are assessable for response. Twenty-two
patients have presented disease progression after the rst cycle. Thirteen
patients remained stable for at least two cycles (12 weeks), two patients
with minimal responses. Toxicities included thrombocytopenia (6 grade 3,
10 grade 4), neutropenia (2 grade 3, 3 grade 4), leukopenia (1 grade 3, 1
grade 4), AST elevation (1 grade 3), ALT elevation (1 grade 3, 1 grade 4),
and infection (1 grade 3). Cloretazine shows very limited disease stabiliza-
tion in recurrent GBM. However, it is too early to determine the response in
recurrent AA/AO. Toxicities are limited to hematologic events and transient
transaminase elevation.
142. CELLULAR PATHWAYS MEDIATING APO2L/TRAIL-
INDUCED APOPTOSIS IN GLIOMA CELLS
J. Rieger, M. Weller, B. Frank, and W. Wick; Laboratory of Molecular
Neuro-Oncology, Department of General Neurology and Hertie-
Institute for Clinical Brain Research, Tuebingen, Germany
Apo2L ligand/TNF-related apoptosis-inducing ligand (APO2L/TRAIL)
is a member of the TNF family that has been shown to induce apoptosis
in malignant cells but not in most normal cells. Moreover, Apo2L/TRAIL
exhibits synergistic effects with irradiation or chemotherapeutic drugs
including lomustine (CCNU) and temozolomide. Previous work showed
that most glioma cell lines are resistant to APO2L/TRAIL-induced apopto-
sis unless co-sensitized by cotreatment with an inhibitor of protein synthesis
such as cycloheximide (CHX). Resistance is also observed in some cell lines
expressing high levels of the agonistic receptors TRAIL-R1 and TRAIL-
R2. However, even in the presence of CHX, some cell lines are still resistant
to APO2L/TRAIL. Further, the mechanism by which CHX renders resis-
tant cell lines sensitive to APO2L/TRAIL has remained largely unclear.
Because PI3K and casein kinase II signaling have been shown to mediate
survival, we investigated whether inhibitors of these signaling paths would
modulate the sensitivity of glioma cell lines to APO2L/TRAIL. We nd
that the inhibition of PI3K by LY294002 is less potent in sensitizing glioma
cell lines to APO2L/TRAIL than the inhibition of casein kinase II by DRB.
In addition, there is a strong correlation between the sensitizing effects of
CHX, LY294002, and DRB. Cell lines that are resistant to APO2L/TRAIL,
even in the presence of CHX, are not rendered sensitive to APO2L/TRAIL
by these inhibitors. This suggests that there are common molecular sur-
vival pathways that are modied by inhibition of protein synthesis (CHX),
PI3K (LY294002) and casein kinase II (DRB), all resulting in sensitivity to
APO2L/TRAIL-induced apoptosis in glioma cells.
143. MATRIX METALLOPROTEINASE
INHIBITORINDUCED JOINT-RELATED TOXICITY
PREDICTS PROLONGED PROGRESSION-FREE SURVIVAL IN
RECURRENT HIGH-GRADE GLIOMA TRIALS
M. Groves, Vinaykumar Puduvalli, Charles A. Conrad, Mark R. Gilbert,
Kenneth R. Hess, W.K. Alfred Yung, and Victor A. Levin; Neuro-
Oncology, The University of Texas M.D. Anderson Cancer Center,
Houston, Texas, USA
Recent reports have highlighted relationships between a drugs toxicity
and its anti-cancer effect. Variability in drug-induced toxicity can be due
to drug metabolism, drug-drug interactions, or pharmacogenomic factors.
We reviewed outcomes for 93 patients treated on 2 prospectively conducted
phase 2 studies evaluating temozolomide (TMZ) (150200 mg/m
2
/day,
days 15) plus the matrix metalloproteinase inhibitor marimastat (MRM)
(25 mg BID days 828) for recurrent glioblastoma multiforme or anaplastic
glioma. Histologic subgroups were analyzed individually and together, look-
ing for the effect of toxicity due to MRM (presence and grade of joint-related
toxicity (JRT)) and its relationship to progression-free survival (PFS). We
reviewed outcomes for all 93 patients, 44 with GBM, 23 with AA, 14 with
AO, 12 with AOA. PFS was signicantly longer in pts with JRT (median 36
weeks vs. 9 weeks, P 0.0001, hazard ratio 0.3). This difference remained
highly signicant after adjustment for clinical factors (age, KPS, extent
resection, histology, prior nitrosourea, enzyme-inducing anti-epileptic
drug [EIAED] status). However, the difference was bigger in patients on
EIAEDs (EIAED: 69 weeks vs. 9 weeks, No EIAED: 24 weeks vs. 9 weeks).
This interaction was signicant after adjustment for the clinical factors (P
0.032), but not prior to adjustment (P 0.20). The adjusted hazard ratio
for JRT is 0.4 in pts without EIAED, and 0.1 in pts with EIAED. There was
a graded difference in PFS with grade of JRT (0: Median PFS 9 weeks,
1: 24 weeks, 2: 33 weeks, 3: 38 weeks). Again these differences were more
dramatic in patients taking EIAED. After adjustment for clinical factors,
this interaction between EIAED use and JRT grade was signicant (P
0.056). The adjusted hazard ratios by grade for patients not taking EIAEDs
are 0: 1.0, 1: 0.7, 2: 0.5, and 3: 0.3, and for patients taking EIAEDs they
are 0: 1.0, 1: 0.5, 2: 0.2, and 3: 0.1. The modifying effect of EIAEDs on
JRT was also seen for CR/PR response: In patients not taking EIAEDs,
CR/PR is 0% for patients with and without JRT, but in patients taking
EIAEDs, 3% of the 29 patients without JRT had CR/PR, while 27% of
the 33 patients with JRT had CR/PR (20% in grade 1, 14% in grade 2,
and 33% in grade 3). For those patients who developed JRT, median time
to its onset was 7.7 weeks (1109). Median time to the development of
the most severe JRT was 15 weeks (1109). This analysis demonstrates
the association of MRM-induced JRT with signicantly prolonged PFS in
patients with recurrent malignant glioma. Patients with JRT on EIAEDs
have further enhancement of PFS. Pharmacokinetic and pharmacogenomic
interactions may be involved. Further trials assessing the efcacy of this
combination and its relationship to EIAEDs, drug pharmacokinetics, and
drug-induced JRT are warranted.
144. TIME TO TUMOR PROGRESSION (TTP) AND QUALITY
OF LIFE (QOL) FOLLOWING PROPYLTHIOURACIL
INDUCTION OF CHEMICAL HYPOTHYROIDISM IN FAILED
MALIGNANT GLIOMAS
E. Linetsky,
1
A. Hercbergs,
2
S. Dotan,
1
E. Shalom,
1
and T. Siegal
1
;

1
Leslie and Michael Gafn Center for Neuro-Oncology, Hadassah
Hebrew University Hospital, Jerusalem, Israel;
2
Department of Radiation
Oncology, Cleveland Clinic Foundation, Cleveland, Ohio, USA
Under hypothyroid conditions, gene expression of several growth fac-
tors and their receptors is downregulated. A previous phase 1/2 clinical
study suggested that high-dose tamoxifen given in association with chemi-
cal induction of HT prolongs survival of pts with recurrent malignant glio-
mas. However, a major drawback of induced HT may be a potential nega-
tive effect of HT on the QOL of these vulnerable pts. Our objective was to
evaluate the toxicity and effect of induced HT on TTP and QOL of pts with
failed malignant gliomas. Propylthiouracil was used to induce HT in 20 pts
(median age 49 years) with failed malignant gliomas (8 GBM, 7 anaplastic
astrocytomas, 5 anaplastic oligoastrocytomas). Tamoxifen (240 mg/kg/d)
was given only to pts who became hypothyroid. Clinical evaluation and
QOL were assessed monthly by using a standard questionnaire. Thyroid
function tests were done weekly until HT was induced (TSH 8) and then
repeated monthly and MRI every 8 weeks. Twelve pts (60%) achieved HT
(median TSH 9.8) within a median time of 3 months (range, 1.56 months).
KPS, age, gender, treatment with enzyme inducing antiepileptic drugs, and
baseline TSH levels did not differ between the 2 groups of pts retrospec-
tively stratied as achieving or not achieving HT. Induction of HT often
caused mild fatigue but no other clinical symptoms of HT. Median TTP
was longer in the HT group (5 months vs. 2.7 months, P 0.002), with 6
months PFS of 33% vs. 0%. Clinical improvement was noted in 8/12 pts
(66%) with HT and led to dose reduction or withdrawal of steroid therapy.
Marked decrease in seizure activity was noted, and 2 pts became seizure
free. MRI showed objective response in 25% of the HT group and stable
disease in 75%. At baseline evaluation, the QOL did not differ between
pts who later achieved HT and those who did not. In the HT group, QOL
improved after 3 months and differed signicantly from QOL of the group
without HT based on categories of weakness (P 0.01), fatigue (P 0.01),
depression (P 0.002), interference with family life (P 0.001), global
QOL (P 0.02), and global health (P 0.004) evaluations. Induction of
HT is associated with signicantly longer median TTP in pts with failed
malignant gliomas. Clinical and objective responses are associated with sig-
nicant improvement in QOL despite the induction of HT. Further studies
are warranted to evaluate the benet of early induction of HT shortly after
diagnosis and prior to tumor recurrence.
145. SURGICAL TREATMENT OF LOW- GRADE GLIOMA
RECURRENCES
L. Bello,
1
F. Acerbi,
1
C. Giussani,
1
J. Casagrande,
2
P. Casagrande,
2

D. Spagnoli,
1
M. Caroli,
1
R. Campanella,
1
G. Tomei,
2
and S.M. Gaini
1
;
1
Neurosurgery, University of Milano, Milano;
2
Neurosurgery, University
of Insubria, Varese; Italy
Treatment of low-grade gliomas recurrences is controversial and con-
sists of a wait-and-see policy or treatment based on surgery, chemo, and/or
radiotherapy. The role of surgery, particularly of repeated surgeries, should
still be dened. We present the results of the surgical treatment of 158
patients with a recurrent low-grade glioma admitted at our Dpts during
the decade 1990 to 2000. They were 68 males and 91 females, age ranging
from 22 to 69 years. Follow-up ranged from 50 to 264 months (median 156
months). Only patients with a low-grade astrocytoma, mixed glioma, or
oligodendroglioma were included. Time to rst recurrence correlated with
extent of surgery at rst operation, tumor removal, and extension of the
tumor toward deep structures. Seventy-eight percent of tumors recurred
in the previous location. At surgery, 42% of patients were symptomatic
for seizures or neurological decits, and in 58% an enlarging mass or an
enhancing lesion on MR scan was documented. Global 50% survival was
126 months. Survival correlated with malignant transformation. Malignant
transformation occurred in 57% of cases, at rst or further surgeries, and
correlated with extent of surgery at rst operation and tumor extension and
volume. Survival and malignant transformation correlated also with extent
of resection at surgery for recurrence removal. Time to further recurrence
correlated with extent of resection. A second surgery or further surgeries
were performed in 39%, 22, and 4.5% of cases. Further surgeries did not
result in an increase of morbidity (0.7% mortality, 11% of transient mor-
bidity). Time to recurrence decreased with the increase in the number of
surgeries (from 72 to 23 months). Percentage of total removal decreased
as well (from 51 to 26%). In case of early recurrence, with no ndings
of malignant transformation, early surgery was associated with a longer
survival than when surgery was postponed and a wait and see policy was
adopted. When a recurrence appeared, the combination of surgery eventu-
ally followed by chemotherapy was associated with a longer survival than
when surgery followed chemotherapy. Awake surgery and cortical and sub-
cortical stimulations reduced morbidity and improved resection of lesions
close to or involving functional motor or language areas of pathways. Sur-
gery and repeated surgeries for low-grade glioma recurrences are safe, con-
trol or relieve symptoms, and may postpone the use of adjuvant therapies
until the time of appearance of malignant transformation.
146. RESPONSE OF RECURRENT PILOCYTIC
ASTROCYTOMA IN ADULTS TO TEMOZOLOMIDE (TMZ)
D. Macdonald; Medical Oncology, London Regional Cancer Centre,
London, Canada
The objective of this study was to evaluate the activity of temozolo-
mide (TMZ) chemotherapy for recurrent pilocytic astrocytoma in adults.
Pilocytic astrocytomas (PA) are WHO grade I tumors that usually occur in
children. Surgical resection is often curative. Incompletely resected PAs are
often treated with radiotherapy (RT). Hypothalamic and optic pathway
PAs in children often respond to carboplatin- or lomustine-based chemo-
therapy, but side effects are common and sometimes serious. There is little
information on chemotherapy in adult PA. TMZ is a new, well-tolerated,
oral chemotherapeutic agent with activity in newly diagnosed and recur-
rent malignant gliomas and in low-grade gliomas. Thus, it was appropriate
to evaluate TMZ in PA. The design of the study was a case series. Two
adults with recurrent PA responded to TMZ. A man (case 1), age 54, had
a cerebellar PA partly resected in 1995 and then received RT. Multifocal
recurrence was biopsy-conrmed in February 2002. He received 12 cycles
of standard oral TMZ (200 mg/m
2
/day 5 days, every 28 days). A partial
response (PR) was seen after 6 cycles and a complete response (CR) after 12
cycles. He remains in CR over 20 months off treatment. A woman (case 2),
age 27, had partial resection of a right temporal PA in 1987. MRI progres-
sion was biopsy-conrmed in January 2000 and treated with RT. Symptom-
atic MRI progression was treated with standard TMZ starting November
2001. A minor response was seen after 9 cycles and a PR after 12 cycles.
She received 24 cycles of TMZ with continuing PR more than 10 months
off treatment. TMZ was well tolerated in both patients. Temozolomide is
active in recurrent PA, producing sustained responses with little toxicity,
but prolonged treatment may be needed. Temozolomide should be evaluated
in newly diagnosed pilocytic astrocytoma.
147. SEQUENTIAL ADMINISTRATION OF TEMOZOLOMIDE
AND FOTEMUSTINE IN DE NOVO GBM PATIENTS
N. Oktar, N. Ozdamar, and T. Dalbasti; Department of Neurosurgery,
Ege University, Faculty of Medicine, Izmir, Turkey
The DNA repair enzyme O6-alkylguanine-DNA alkyl transferase (AT)
mediates resistance to chloroethylnitrosoureas. Agents depleting AT such
as DTIC and its new analogue temozolomide (TMZ) can reverse resistance
to chloroethylnitrosoureas. There are some reports concerning the pharma-
cokinetics and sequential administration of temozolomide and fotemustine
treatment in malignant melanoma and malignant glioma patients. In this
clinical study response to sequential administration of p.o. TMZ and infu-
sional fotemustine was evaluated in de novo GBM patients after irradia-
tion. Forty patients with primary glioblastoma multiforme were included in
this study. In group I, ten patients were treated with surgery and radiation
therapy alone. In group II, ten patients received 200 mg/m
2
TMZ for 5
days in 26-day intervals for 6 months. In group III, ten patients received
i.v. 100 mg/m
2
fotemustine every week for 3 weeks and then in three-week
intervals. In group IV, ten patients were treated with a 200-mg/m
2
oral
dose of TMZ for 5 days, and fotemustine in a dose of 100 mg/m
2
was given
intravenously on day 15, one week after TMZ, for six months. Complete
and partial response rates were evaluated for six months both with MRI
and with neurological evaluation. Time to tumor progression, Ki67 %,
Karnofsky performance status, and survival rates for each group were cal-
culated. Combination therapy of two agents were well tolerated in 80% of
the patients without any signicant side effects. Signicant although statis-
tically not proven better prognostic results, even a complete response, were
achieved in the sequential administration of TMZ and fotemustine. Other
ways of administration that are discussed, such as i.v. TMZ or locoregional
administration, have yet to be established.
148. DO WE NEED A MORE DIFFERENTIATED APPROACH
FOR THE TREATMENT OF NEUROCYTOMAS?
D. Rades,
1
F. Fehlauer,
1
K. Lamszus,
2
C. Hagel,
3
and S.E. Schild
4
;
1
Radiation Oncology,
2
Neurosurgery, and
3
Neuropathology, University
Hospital Hamburg-Eppendorf, Hamburg, Germany;
4
Radiation
Oncology, Mayo Clinic Scottsdale, Scottsdale, Arizona, USA
Reports on neurocytoma became more frequent after 1995, which
reects the increasing importance of this entity. This analysis aims to give
treatment recommendations for well-differentiated neurocytomas, atypi-
cal neurocytomas (MIB-1 index 3%, atypical histology), and neurocyto-
mas in children. All reported neurocytoma cases and our previous papers
were reviewed for age, gender, histology, MIB-1-labeling index, extent of
surgery, radiotherapy (RT), local control (LC), and survival (OS). More
data were obtained directly from the authors. This approach provided more
detailed information and a longer follow-up period than the data from the
literature alone. Complete resection (CTR), CTR RT, incomplete resec-
tion (ITR), and ITR RT were compared for LC and OS at 5 years and
at 10 years. Comparisons were performed for the whole series, for well-
differentiated lesions, for atypical lesions, and for neurocytomas in children
18 years. In the ITR RT group, radiation doses 54Gy and 54 Gy were
compared. Data were complete in 427 patients (70 children, 357 adults);
321 patients had well-differentiated lesions, and 86 had atypical lesions.
Well-differentiated lesions were associated with better LC and OS than
atypical lesions (P 0.001). CTR was signicantly superior to ITR (P
0.001). After CTR, outcome was not signicantly improved by RT. After
ITR, RT improved OS in the whole series (P 0.04), in patients with well-
differentiated lesions (P 0.03), and in patients with atypical lesions (P
0.05), but not in children (P 0.19). LC was signicantly improved in all
groups (P 0.001, children P 0.01). A dose 54 Gy appeared benecial
only after ITR of atypical lesions (P 0.05 for LC). In children, 50 Gy
and 50Gy appeared comparably effective for LC and OS. CTR should be
performed whenever possible and does not require postoperative RT. After
ITR, RT improves outcome. The 54-Gy dose appears sufcient for long-
term control of well-differentiated lesions; 5054 Gy appears sufcient for
children; 5660 Gy is required for atypical lesions, depending on the treat-
ment volume and the expected toxicity.
149. CONCURRENT ADMINISTRATION OF PHENYTOIN
DOES NOT AFFECT THE PHARMACOKINETICS OF
CELECOXIB IN PATIENTS WITH NEWLY DIAGNOSED
GLIOBLASTOMA MULTIFORME
S. Grossman, J. Olson, T. Batchelor, D. Peereboom, G. Lesser, S.
Desideri, X. Ye, T. Hammour, and J. Supko; NABTT CNS Consortium,
Baltimore, Maryland, USA
Evidence implicating cyclooxygenase-2 (Cox-2) in tumor angiogenesis
has generated interest in evaluating the role of inhibitors of this enzyme,
such as celecoxib, in cancer treatment. Hepatic metabolism by cytochrome
P450 2C9 (CYP2C9) is a major route of elimination for celecoxib. Anti-
epileptic drugs that are frequently used in glioblastoma patients induce a
number of CYP450 enzymes, including CYP2C9. This study was conducted
to determine the effects of enzyme-inducing antiepileptic drugs (EIAEDs)
on the pharmacokinetics of celecoxib. Secondary objectives were to deter-
mine the safety of celecoxib in this setting and estimate the duration of sur-
vival when celecoxib was administered concurrently with radiation therapy
in patients with newly diagnosed glioblastoma multiforme. Patients were
divided into 2 groups (EIAED and EIAED) based on anticonvulsant use.
Celecoxib administration began one week before conventional radiation
therapy was started. A single 400-mg dose of celecoxib was taken on the
rst day of treatment. Twice-daily dosing (400 mg every 12 h) was begun
the following day and continued until tumor progression, dose-limiting tox-
icity, or study withdrawal. No adjuvant chemotherapy was permitted. Phar-
macokinetic blood samples were obtained serially for 24 h after the rst
dose of celecoxib and prior to a morning dose once a week during weeks 2
to 6. A validated LC/MS assay was used to determine the concentration of
celecoxib in plasma. A total of 35 patients (22 EIAED and 13 EIAED)
were accrued from October 2003 to September 2004. Fourteen patients
(40%) were women, and 5 (14%) were African American. All patients in
the EIAED group were receiving phenytoin. The study was closed before
reaching the stated goal of 22 patients in each cohort in light of the posi-
tive results of the EORTC adjuvant temozolomide study. Celecoxib therapy
was very well tolerated, and no gastrointestinal bleeding or renal toxicities
were noted. Only one patient complained of grade 34 epigastric distress
and this responded to a dose reduction. Pharmacokinetic data is currently
available for 14 EIAED patients and 10 EIAED patients. There was no
signicant difference (P 0.66) between the maximum concentration of
drug in plasma following administration of the rst dose in the EIAED
(1.85 0.71 g/ml) and EIAED (1.98 0.48 g/ml) groups. Similarly,
the area under the plasma concentration-time prole from time zero to 24
h was similar (P 0.79) for the two groups (EIAED, 14.9 5.7 gh/
ml; EIAED, 13.9 9.1 gh/ml). Survival gures remain too premature
to report, as 31 of the 35 patients remain alive. These preliminary results
strongly suggest that the plasma pharmacokinetics of celecoxib is not sig-
nicantly affected by the concomitant administration of EIAEDs. Further-
more, plasma levels in this study are similar to data in published reports on
patients with arthritis who were not receiving concomitant glucocorticoids.
This drug was well tolerated in this clinical setting. Survival results will be
presented as the data matures.
150. A MONO-INSTITUTIONAL, PHASE 2, DOSE-
ESCALATION STUDY OF SAFETY/EFFICACY OF NEWER,
RECENTLY MARKETED, ANTICONVULSANT DRUGS FOR
TUMOR-ASSOCIATED EPILEPSY (TAE)
M. Riva, A. Gatti, F. Imbesi, G. DAliberti, A. La Camera, C.A. Defanti,
and M. Collice; Ospedale Niguarda, NeuroSciences, Milano, Italy
Seizures are a common symptom of brain tumors and have a signicant
impact on neurobehavioral functioning and quality of life (QoL). Patients
with seizures are often fearful of having them and may become socially
isolated. Old anticonvulsant drugs (AEDs) - such as phenytoin (PHT), phe-
nobarbital (PB), and carbamazepine (CBZ) induce the CYP450 enzyme
system (interfering with other commonly used drugs and increasing chemo-
therapeutic agent clearance) and produce, in these pts, more idiosyncratic
and side effects than in a general epilepsy-pt population. Furthermore, one
third of these pts could be dened as drug-resistant, and only one third
achieves successful seizure control. At present, physician practice in TAE
prophylaxis is characterized by signicant behavioral heterogeneity, and
literature lacks data concerning efcacy and toxicity of new, recently mar-
keted, AEDs. A monoinstitutional phase 2 study has been carried out to
compare monotherapy efcacy (topiramate, lamotrigine, and oxcarbaze-
pine) in a dose-escalation study for TAE. The characteristics of the three
groups are as follows: (1) lamotrigine group: 33 primary and 7 metastatic
brain tumor patients (P-MBT); 52% epilepsy as symptomatic onset, 48% in
the follow-up; treatment (median) 6 months; efcacy in 62%; toxicity 4 pts:
1 Stevens-Johnson, 3 intense skin reaction; (2) topiramate group: 38 PBT
and 14 MBT; 44% epilepsy as symptomatic onset, 56% in the follow-up;
treatment (median) 8 months; efcacy in 76%; no toxicity; (3) oxcarbaze-
pine group: 49 PBT, 11 MBT; 57% epilepsy as symptomatic onset, 43% in
the follow-up; treatment (median) 8 months; efcacy in 71%; toxicity 7 pts:
5 hypo-Na
, 2 intense skin reaction. We conclude that in the present study,

LTG, TPM and Ox-CBZ have shown the following, even in monotherapy:
(1) a clinical efcacy in seizure control similar to the one obtained with
old-AEDs: 30.3% of pts suffered from one or more Ps and/or GTCS in
the whole follow-up, requiring polytherapy in about 10% (better for TPM
and OxCBZ than LMT); (2) fewer side effects (especially for TPM); (3) no
signicant pharmacological interferences; and (4) no need for hematologi-
cal controls. Some general considerations are possible: (1) Br Mets-TAE is
more responsive to treatment than high-grade glioma-TAE (75 vs. 64%);
(2) plasma levels of the new AEDs do not correlate with clinical control;
(3) maximum tolerated doses seem to be advisable and dose-increase often
results in a satisfactory efcacy after a seizure. However, the most relevant
practical problems in neurooncological pts treated with 2nd-generation
AEDs are (1) the necessity of a long period of titration, to avoid side effects
and (2) the only possibility of an oral administration. In conclusion, new
AEDs have proved to be useful and handy drugs in the management of a
difcult epilepsy such as TAE.
151. TEMOZOLOMIDE IN GLIOMATOSIS CEREBRI
E. Laguzzi,
1
R. Rud,
1
F. Giunta,
1
A. Pace,
1
M. Salvati,
1
M. Scerrati,
1

A. Silvani,
1
C. Carapella,
1
and R. Sofetti
1
; for the Italian Association of
Neuro-Oncology (AINO, Italy)
Gliomatosis cerebri is a diffusely growing neuroepithelial tumor whose
optimal treatment is unclear. Few data are available about response to radi-
otherapy and chemotherapy. The objective of this prospective study was to
assess the efcacy of temozolomide in patients with gliomatosis cerebri.
Since 1999, 28 patients with histologically conrmed (biopsy or partial
resection) gliomatosis cerebri were treated with temozolomide either at pro-
gression after prior radiotherapy/chemotherapy or upfront. Tissue speci-
mens were diagnostic for glioblastoma in 2 cases, malignant glioma in 5,
anaplastic astrocytoma in 4, gemistocytic astrocytoma in 2, astrocytoma in
9, oligoastrocytoma in 1, oligodendroglioma in 2, glial proliferation typical
of gliomatosis cerebri in 3. Patient characteristics were as follows: median
age, 46 years (range, 1470 years); 13 males and 15 females; median KPS
at diagnosis 70 (range, 5090). Presenting symptoms were as follows: sei-
zures (11 patients), intracranial hypertension (7), motor decits (5), mental
status changes (2), drowsiness and diplopia (2), dizziness and vomiting
(1). Twelve out of 28 pretreatment MRI scans demonstrated some contrast
enhancement. Eleven of 28 patients had received radiation therapy, and 4
had received chemotherapy (BCNU) prior to temozolomide. All patients
were treated with temozolomide, 200 mg/m
2
per day for 5 days every 4
weeks until progression or unacceptable toxicity. Response was evaluated,
according to Macdonald criteria, on MRI using both T1-weighted with
gadolinium and FLAIR images. The median number of cycles was 7 (range,
120). One patient (4%) showed a CR of the contrast-enhancing area, 2
patients (7%) a PR of the Flair hyperintense area, 14 (50%) an SD, and 11
(39%) a PD. Among patients with SD, 2 had a reduction of tumor volume
of 20% to 40% (minor response). Overall response rate (CR PR
minor response) was 18%. Median time to tumor progression (TTP)
was 6 months (range, 127), with a median survival of 12 months (range,
3119). A clinical benet, consisting in a reduction of seizures or improve-
ment of intracranial hypertension, was observed in 9/28 patients (32%).
PFS at 6 months was 57%, at 12 months 25%. Three patients showed grade
IIIIV hematological toxicity. Temozolomide seems to be effective in gli-
omatosis cerebri. The study is ongoing.
152. TEMOZOLOMIDE (TMZ) WITH O6-BENZYLGUANINE
(BG) PLUS CPT-11 IN THE TREATMENT OF RECURRENT
MALIGNANT GLIOMA: A PHASE I TRIAL
A. Desjardins, J. Quinn, D. Reardon, J. Rich, S. Sathornsumetee, J.
Vredenburgh, A. Walker, S. Tourt-Uhlig, D. Bigner, and H. Friedman;
Duke University Medical Center, Durham, North Carolina, USA
The combination of TMZ and CPT-11 reveals a marked increase in
activity compared with either agent used alone. The addition of BG to this
combination dramatically increased the growth delay of the O6-alkylgua-
nine-DNA alkyltransferase (AGT)-positive malignant glioma D-456MG.
Those results have prompted the initiation of a phase 1 study of TMZ with
BG plus CPT-11 in patients with malignant glioma. Eligibility included
adult patients with recurrent malignant gliomas. Patients were divided in
two strata: those receiving enzyme-inducing anticonvulsant (EIAC) and
those not receiving EIAC. Each patient was treated with a 1-h BG infusion
at 120 mg/m, followed by a single dose of TMZ at 355 mg/m and a 90-
min infusion of CPT-11 in dose escalation (starting at 60 and 40 mg/m,
respectively), and thereafter a 48-h continuous BG infusion at 30 mg/m/
day. Repeat dosing of TMZ and CPT-11 occurs on day 1 of a 21-day cycle.
Responses were assessed by functional and imaging criteria after two cycles
(6 weeks). The primary end points of this study were to determine the maxi-
mum tolerated dose (MTD) of CPT-11 when administered following TMZ
plus BG and to determine the activity and toxicity of this combination. Fifty
patients have been treated, 47 with glioblastoma multiforme (GBM), two
with anaplastic astrocytoma (AA), and one with anaplastic oligodendro-
glioma (AO). Twenty-two patients have been enrolled to the EIAC stratum
at CPT-11 doses of 60, 90, 120, and 150 mg/m. Twenty-eight patients have
been accrued to the non-EIAC stratum at CPT-11 doses of 40, 60, and 80
mg/m. In the non-EIAC stratum, dose de-escalation for TMZ was required
from 355 to 267 and then to the present dose of 200 mg/m. Dose-limiting
toxicities observed thus far have been limited to hematologic toxicities,
including neutropenia (9 grade 4), leukopenia (1 grade 4), and thrombo-
cytopenia (2 grade 4). Forty-one patients are assessable for response. One
patient continues to show a partial response after 10 cycles, 14 showed a
stable disease for at least 4 cycles, and 25 patients progressed after either
the rst or second cycle. The MTD of this drug combination for the non-
EIAC stratum has been dened at a dose of TMZ of 200 mg/m and a dose
of CPT-11 of 60 mg/m with DLT limited to hematologic events. The MTD
has yet to be identied for the EIAC stratum. Partial response has been
observed in one patient, which remains on treatment after 10 cycles.
153. A COMPARISON OF RECOVERY FOLLOWING
REMIFENTANIL-DESFLURANE AND REMIFENTANIL-
SEVOFLURANE ANESTHESIA FOR PATIENTS UNDERGOING
CRANIOTOMY FOR TUMOR
M. Varma,
1
S. Williamson,
1
and P. Kane
2
; Departments of
1
Anaesthesia
and
2
Neurosurgery, James Cook University Hospital, Middlesborough,
UK
Craniotomy for tumor carries the risk of postoperative complications
such as bleeding, cerebral edema, and cerebral ischemia. If not diagnosed
early and treated promptly, these can lead to neurological decit. There-
fore, rapid recovery and early neurological assessment are useful goals in
the anesthetic management of patients undergoing craniotomy for tumor.
The anesthetic technique should enable a rapid and predictable recovery. The
pharmacology of remifentanil and desurane suggests that recovery will
be faster if used in combination compared to remifentanil/sevoflurane
anesthesia. We compared emergence from remifentanil/desurane versus
remifentanil/sevourane anesthesia in patients undergoing craniotomy for
tumor. Forty patients undergoing anesthesia for elective craniotomy for
tumor were randomly assigned to receive remifentanil/desurane or remi-
fentanil/sevourane anesthesia. Following induction with remifentanil,
propofol and rocuronium, anesthesia was maintained with study vapor and
remifentanil in oxygen and air. All treatment was standardized. Recovery
staff blinded to the study recorded early recovery parameters. The times
required for spontaneous ventilation, eye opening, extubation, stating
name, stating date of birth and achieving post anesthesia recovery score
(Aldrete) 9 were 50% shorter after remifentanil/desurane compared to
remifentanil/sevourane anesthesia. In patients undergoing craniotomy for
tumor surgery, recovery is signicantly faster and more predictable after
remifentanil/desurane compared to remifentanil/sevourane anesthesia
allowing an earlier neurological examination.
154. A PHASE 1 TRIAL OF OSI-774 (TARCEVA) IN PATIENTS
(PTS) WITH RECURRENT MALIGNANT GLIOMAS (MG)
ON ENZYME INDUCING ANTI- CONVULSANTS: A NORTH
AMERICAN BRAIN TUMOR CONSORTIUM TRIAL
J. Raizer,
1
L. Abrey,
2
P. Wen,
3
T. Cloughesy,
4
I.A. Robins,
5
H.A.
Fine,
6
F. Lieberman,
7
V.K. Puduvalli,
8
K.L. Fink,
9
and M. Prados
10
;
1
Northwestern University, Feinberg School of Medicine, Chicago,
Illinois;
2
Memorial Sloan-Kettering Cancer Center, New York, New
York;
3
Dana-Farber Cancer Institute, Boston, Massachusetts;
4
University
of California, Los Angeles, Los Angeles, California;
5
University of
Wisconsin Hospital & Clinics, Madison, Wisconsin;
6
Neuro-Oncology
Branch, NCI, NIH, Bethesda, Maryland;
7
University of Pittsburgh,
Pittsburgh, Pennsylvania;
8
The University of Texas M.D. Anderson
Cancer Center, Houston, Texas;
9
The University of Texas Southwestern
Medical Center, Dallas, Texas;
10
University of California San Francisco,
San Francisco, California; USA
To determine the MTD of OSI-774, a small molecule tyrosine kinase
inhibitor of the epidermal growth factor receptor (EGFR), in patients with
recurrent malignant gliomas on enzyme inducing anti-convulsant drugs
(EIAEDs). Pts with recurrent MG or meningioma were treated in a stan-
dard phase 1 design with 3 pts per cohort until the DLT was reached. DLT
was dened as grade 3 thrombocytopenia, grade 3 or 4 anemia or neu-
tropenia, any grade 3 nonhematologic toxicity, or failure to recover from
toxicities within 2 weeks of the last dose of OSI-774 treatment rst cycle.
The starting dose was 150 mg/day continuously and increased by 50 mg
for cohort 2, 75 mg for cohort 3, and then in 125-mg increments for all
subsequent cohorts until the DLT was reached. Patients could not have had
more than 3 prior relapses and 2 prior chemotherapies. Patients were evalu-
ated for response with MRI every 56 days. PK studies and AGP levels were
done in most patients. Thirty-two pts were enrolled: 21 GBM (14M:7F), 8
AA (4M:4F), 2 AO (Male), and 1 atypical meningioma (Female). Median
age was 44 (1976), and median KPS was 90 (60100). Number of prior
chemotherapies was as follows: 2 pts had none, 11 pts had 1, 12 pts had
2, and 3 pts had 3. The DLT occurred at 775 mg/day and consisted of one
each of the following: grade 3 rash, grade 3 hypophosphatemia, and grade 3
thrombocytopenia. The MTD was determined to be 650 mg/day; 1 patient
out of 6 patients had DLT at that level (grade 3 deep vein thrombosis/
pulmonary embolism). The maximal tolerated dose of OSI-774 for patients
on EIAEDs is 650 mg/day. Pharmacokinetic data and outcome data will
be presented.
155. CONTRIBUTION OF RADIOSURGERY IN THE
SURGICAL TREATMENT OF SKULL BASE MENINGIOMA
Y. Sonoda, S. Takemura, K. Sakurada, M. Saino, S. Sato, S. Saito,
H. Jokura, and T. Kayama; Department of Neurosurgery, Yamagata
University School of Medicine, Yamagata, Japan
Management of skull base meningiomas has been challenging, even
though they are benign tumors. Since tumors are located deep in the skull
and can involve important neurovascular structures, surgical treatment has
been associated with high morbidity and mortality. However, advances in
surgical instrumentation and newly developed approaches to the cranial
base were introduced. Simultaneously, radiosurgery was introduced to treat
skull base tumors. With this combination of modern techniques, we would
like to discuss how radiosurgery contributes to the treatment of skull base
meningiomas. During 1995 to 2004, 50 patients with skull base meningio-
mas underwent surgical treatment in our department. The mean patient age
was 56 years (range, 2272 years). Sixteen patients (32%) were men, and
34 (68%) were women. The tumors were located at anterior cranial base
(n 26: 3 olfactory groove, 2 planum sphenoidale, 10 tuberculum sellae,
and 10 anterior clinoidal process), middle cranial base (n 4: 4 cavern-
ous sinus), and posterior cranial base (n 20: 12 petroclival, 7 petrous
apex, 1 foramen magnum). Nineteen patients (38%) had progressive visual
impairment. Cranial nerve palsy was found in 9 cases (18%). Nine patients
(18%) showed conscious disturbance due to increased intracranial pressure.
Our treatment strategies are (1) the tumor should be left undetached in
case there is no CSF space between the tumor and the surrounding struc-
tures to avoid new decit, (2) the residual volume should be reduced to
less than 20 ml, which is small enough for radiosurgery (3) the distance
between residual tumor and optic nerve should be less than 3 mm. Total
removal was achieved in 33 cases (66%). In the remaining 17 (34%), a small
amount of tumor was intentionally left because of invasion of the cavernous
sinus (8 cases), brain stem (4 cases), the perforators from carotid artery or
middle cerebral artery (3 cases), the lower cranial nerves (one case), or the
optic nerve (one case). Postoperative mortality was none and morbidity
was found in 6 cases (12%). Visual function was improved in 16 patients
(89%). The radiosurgery was followed within three months after the open
surgery in 14 patients. During the follow-up period (5112 months; mean,
59 months), tumor regression was observed in 7 patients (50%), and the
tumor was unchanged or decreased in 7 patients. The tumor growth con-
trol rate was 100%. No patients experienced deterioration of their clinical
symptoms after radiosurgery. In the majority of the cases, we could totally
remove the tumors safely. On the other hand, we left a small part of the
tumors invading important neurovasucular structures and treated residual
tumors by radiosurgery. Radiosurgery was a safe and effective treatment
for residual tumors. Our result indicated radiosurgery could change surgi-
cal strategy of skull base meningiomas invading important neurovasucular
structures.
156. PHASE 1/2 TRIAL OF A TWICE-DAILY REGIMEN OF
TEMOZOLOMIDE AND CELECOXIB FOR TREATMENT OF
RELAPSED/REFRACTORY GLIOBLASTOMA MULTIFORME
AND ANAPLASTIC ASTROCYTOMA
S. Pannullo,
1
J. Burton,
2
J. Serventi,
1
P. Stieg,
1
S. Hariharan,
3

R. Elsoueidi,
2
I. El-Jassous,
2
and C. Balmaceda
4
;
1
Neurological Surgery,
New York Presbyterian Hospital-Weill Cornell, New York, New York;
2
Medicine, Staten Island University Hospital, Staten Island, New York;
3
Neuroscience, JFK Medical Center, Edison, New York;
4
Neurology,
New York Presbyterian Hospital-Columbia University, New York, New
York; USA
The COX-2 enzyme is overexpressed in anaplastic astrocytoma (AA)
and glioblastoma (GBM), and COX-2 inhibitors have preclinical efcacy in
glioma models. Since this drug class has nonoverlapping toxicity with che-
motherapy, a phase 1/2 clinical trial was initiated for patients with recur-
rent or progressive AA or GBM with the combination of temozolomide
(TMZ) and celecoxib (CEL). For phase 1, a modied Fibonacci design was
used with 3 patients per cohort. Chemotherapy was xed, with TMZ being
given with an oral loading dose of 200 mg/m
2
followed by 9 doses of 90
mg/m
2
BID for 5 days. CEL was given in 5 escalating dose levels starting
at 60 mg/m
2
BID up to 240 mg/m
2
BID (maximum of 400 mg BID) for 10
days. Cycles were repeated every 28 days. Forty-six patients (28 M, 18 F)
received 235 cycles of therapy, with 37 of the patients carrying a diagnosis
of GBM and 9 with AA. Prior treatment consisted of radiation (N 46)
and chemotherapy (N 12). Median age was 54 years (range, 3474).
This BID regimen of TMZ plus CEL was well tolerated by most patients,
and no dose-limiting toxicity was observed at any of the 5 CEL dose levels.
Hematologic toxicity was mild with grade 4 neutropenia occurring in 1/235
(0.4%) cycles, grade 3 neutropenia in 2/235 (.85%), and grade 3 thrombo-
cytopenia in 3/235 (1.3%). Grade 3/4 toxicity did not recur following TMZ
dose reduction. Grade 1/2 constipation was common, occurring in 13/46
(28%) patients. Tumor responses were evaluated every 8 weeks. In the 41
patients evaluable for response, after 2 cycles, 3/41 pts (7.4%) had a partial
response (PR), 32/41 (78%) had stable disease (SD), and 6/41 (14.6%) had
progressive disease (PD), resulting in an overall response rate (OR) of 85%.
After 6 cycles, the responses were as follows: 1/18 (5.6%) CR, 5/18 (27.8%)
PR, 5/18 (27.8%) SD, and 7/18 (38.9%) PD, for an OR of 61%. One patient
had a PR in the brain for 13 cycles, but developed tumor in the cervical spi-
nal cord. The average duration of response was 5.6 months (range, 215).
The 6-month progression-free survival rate for the 41 evaluable patients
was 13/41 (31.7%), with a 6-month overall survival rate of 31/41 (75.6%).
A regimen of twice-daily TMZ and CEL is safe and potentially effective for
the treatment of recurrent high-grade gliomas. Further study of TMZ plus
CEL-based regimens is warranted.
157. LONG-TERM SURVIVAL WITH BLOOD-BRAIN
BARRIER DISRUPTION THERAPY IN PATIENTS WITH
GLIOBLASTOMA MULTIFORME: A REPORT OF THREE
CASES
H. Ulrich-Pur,
1
S. Hofmann,
2
M. Hassler,
1
K. Heimberger,
3
B. Fazeny-
Drner,
1
K. Dieckmann,
4
C. Dittrich,
6
E. Suess,
5
and E. Marosi
1
;
1
Clinical Oncology/Med I and
2
Departments of Internal Medicine IV,
3
Neuroradiology,
4
Radio-oncology, and
5
Neurology, Medical University
Vienna, Vienna;
6
Clinical Division of Oncology, Department of Internal
Medicine III, Kaiser Franz Josef-Hospital Vienna, Vienna; Austria
Long-term survival is rare in patients with glioblastoma multiforme
(GBM). The 5-year survival rate has remained at 2% for the last 30 years.
One of the problems of glioblastoma chemotherapy is the penetration of
drugs through the blood-brain barrier (BBB). BBB disruption therapy pro-
tocols have been established and are mainly used in the therapy of primary
central nervous system lymphoma. We present three cases of highly malig-
nant gliomas who became long-term survivors (LTGBMS) after blood-
brain barrier disruption therapy (BBBD). After debulking surgery, three
patients with high-grade gliomas underwent a blood-brain barrier disrup-
tion (BBBD) therapy consisting of a 30-s intracarotid infusion of mannitol
followed by both intraarterial and intravenous chemotherapy. Cytotoxic
regimen consisted of intraarterial methotrexate 1200 mg on days 1 and 2,
intravenous cyclophosphamide 15 mg/kg on days 1 and 2, and intravenous
etoposide 150 mg/m
2
on days 1 and 2. Treatment courses were repeated
every 4 weeks for a total of 4 courses. Noteworthy, two of the patients had
no radiotherapy after surgery. Cranial magnetic resonance imaging follow-
up was performed every 3 to 6 months until progression occurred, 6 years,
10 years, and 11 years and 11 months after initial diagnosis, respectively.
At progression, patients received a second-line intravenous chemotherapy
consisting of fotemustine in combination with dacarbacine. Despite the use
of cytotoxic drugs that were of almost marginal benet when used intrave-
nously in patients with high-grade gliomas, after BBBD, patients survived
for 8 years, 12.9 years, and 13.4 years, respectively. These patients had the
benet of periods of disease-free survival lasting 6 years, 10.9 years, and
11.9 years. Our data suggest that in selected cases of high-grade glioma
patients, BBBD therapy is effective and leads to long periods of disease-free
survival. Unfortunately, even more than ten years after therapy, patients
relapsed near the initial tumor site, highlighting the need for further thera-
peutic progress in GBM therapy.
158. TEN CASES OF SYMPTOMATIC SINGLE
PARENCHYMAL CNS METASTASES TO THE PINEAL
GLAND FROM SYSTEMIC MALIGNANCIES
B. Lassman,
1
J.N. Bruce,
2
and M.R. Fetell
3
;
1
Neurology, Memorial
Sloan-Kettering Cancer Center, New York, New York;
2
Neurological
Surgery and
3
Neurology, Columbia University Medical Center, New
York, New York; USA
Metastases to the pineal gland are typically noted as incidental nd-
ings at autopsy. In contrast, we identied 10 cases of symptomatic single
parenchymal metastases to the pineal gland. This is the largest antemortem
series reported. Lung cancer was the primary tumor in 3 cases (2 non-small
cell cases, 1 small cell case). Breast, renal, cervical, esophageal, gastric,
and colon cancers were the primary tumor type for 1 case each, as was a
cancer of unknown primary. In 4 cases, the neurologic symptoms antedated
the discovery of a systemic malignancy; in the other 6 cases, the primary
tumor was in remission when neurologic symptoms developed. There were
6 women and 4 men with a median age of 56 years (range, 3670). The
clinical and radiographic ndings were fairly uniform: In all cases, a con-
trast-enhancing pineal lesion with obstructive hydrocephalus was discov-
ered by cranial CT or MRI, and the pineal metastasis produced the rst
clinical manifestation of CNS disease. The metastasis or associated hydro-
cephalus induced mental status changes ranging from confusion to coma
in 8 patients. Oculomotor or pupillary abnormalities were observed in 6
patients including features of Parinauds or Sylvian aqueduct syndromes.
Documented leptomeningeal tumor spread was present at the time of diag-
nosis in 6 patients. Treatment of hydrocephalus by CSF diversion (with
ventriculoperitoneal shunting or third ventriculostomy in 4 cases each) led
to transient clinical improvement in 7 patients and no change in 1. Radio-
therapy was given to 6 patients with at least partial response in 4. However,
the overall prognosis was poor, with median survival of 4.5 months (range,
192) from the time of rst diagnosis of pineal metastasis. Only 2 patients
survived beyond 1 year (21 months and 92 months), neither of whom had
leptomeningeal spread. Metastatic disease should be considered in the dif-
ferential diagnosis of pineal tumors even in the absence of a history of sys-
temic cancer. Leptomeningeal spread is an important feature that should be
investigated in cases of metastases to the pineal gland.
159. PRODIGE: A PHASE 3 RANDOMIZED PLACEBO-
CONTROLLED TRIAL OF THROMBOPROPHYLAXIS USING
DALTEPARIN LOW-MOLECULAR-WEIGHT HEPARIN
(LMWH) IN PATIENTS WITH NEWLY DIAGNOSED
MALIGNANT GLIOMA
J. Perry, L. Rogers, N. Laperriere, J. Julian, W. Geerts, G. Agnelli, M.
Malkin, R. Sawaya, R. Baker, T. Finch, and M. Levine; Ontario Clinical
Oncology Group and PRODIGE Investigators. Hamilton, Ontario,
Canada
Venous thromboembolism (VTE) is common in patients with malig-
nant glioma, occurring in 20% to 30% of patients per year of survival.
Clinical risk factors may include tumor grade, leg weakness, the use of
chemotherapy, and surgery. Recently it has been shown that Tissue Factor
(TF), the principal initiator of coagulation, is overexpressed in malignant
glioma cell lines and human tumor samples. TF may induce a thrombogenic
state in patients and may be a therapeutic target. In addition to antico-
agulant properties, LMWH has been associated with prolonged survival
in patients with nonmetastatic solid tumors through an anticancer effect.
We are conducting a large phase 3 RCT testing the efcacy and safety of
chronic daily administration of dalteparin in patients with newly diagnosed
malignant glioma. Patients are randomized 1:1 to receive dalteparin 5000
anti-Xa units s.c. daily versus daily s.c. placebo. The primary outcome is
VTE-free survival at 6 months, and progression-free survival, overall sur-
vival, toxicity, and neurocognitive performance are secondary outcome
measures. The expected cumulative risk of VTE in the control group is
13% at 6 months. At least 40 events will be needed in order to detect a 60%
relative risk reduction in the dalteparin group with 80% power. Allow-
ing for dropouts, the projected sample size is 512 patients. Clinical risk
factors, concurrent therapies, tumor response, and survival data are being
collected. A companion molecular study examining the role of TF and other
regulators of coagulation is underway. Patients must be adults with newly
diagnosed glioblastoma multiforme or anaplastic glioma, with no history of
prior VTE, and not on chronic anticoagulation. As of November 2004, 124
were patients randomized across 15 active study centers. Of 281 eligible
patients, 157 (56%) were excluded for reasons including conicting trials,
needle aversion, concomitant illness, and possible VTE. An independent
DSMB is supervising this trial and an update on trial progress will be pre-
sented. A placebo-controlled trial testing the role of thromboprophylaxis
in patients with malignant glioma is feasible and is accruing patients at an
increasing number of clinical trial sites worldwide. Of particular interest to
neuro-oncologists is not only the prevention of symptomatic VTE, but the
potential survival advantage conferred by LMWH in other solid tumors.
This study is supported in part by a grant in aid from Pzer Inc. and coor-
dinated by the Ontario Clinical Oncology Group.
160. LONG SURVIVAL RESULTS WITH REPEATED
BIOCHEMOTHERAPY (BUCLADESINE FOTEMUSTINE)
IN DE NOVO GBM PATIENTS
N. Oktar, T. Dalbasti, S. Islekel, and N. Ozdamar; Faculty of Medicine,
Department of Neurosurgery, Ege University, Izmir, Turkey
This clinical study was designed to evaluate the impact of biochemo-
therapy (bucladesine-polymer locoregional therapy and systemic fotemus-
tine chemotherapy) at the time of recurrence of the de novo glioblastoma
multiforme (GBM) patients. In a randomized prospective manner, 50
patients who were diagnosed as de novo GBM were included in this study.
Five different therapy protocols were used. The rst group of 10 patients
had tumor resection only. The second group assessed having only systemic
chemotherapy as six i.v. infusions of fotemustine after tumor resection. The
third group had implantation of bucladesine-loaded biodegradable poly-
meric sustained-release (bcl-SR) pellets. The fourth group received six i.v.
infusions of systemic fotemustine as in the second group, in addition to
local implantation of bcl-SR pellets. Finally, the fth group assessed having
stereotactic biopsy and implantation of bcl-SR rods under local anesthesia
repeated in six-month intervals. In this trial of local interstitial biologic
therapy with long acting (4 to 5 months of release time) bcl-SR did show
a statistically signicant delay of recurrence on the treatment of GBM
patients. The best treatment results were obtained from the local bcl-SR
systemic fotemustinetreated group, in which the survival rate estimated by
Kaplan-Meier method was 100% in de novo GBM at 12 months. Moreover,
in the last group, the mean survival rate was 85.1 25.9 weeks, and 1-year,
2-year, and 3-year survival rates were 100%, 40%, and 10% respectively.
Mean survival times of 28, 32, 37, 63, and 85 weeks for the control, fote-
mustine, bcl-SR, fotemustinebcl-SR and repeated stereotactic bcl-SR
fotemustine groups were achieved, respectively. In this prospective clinical
study conducted on primary GBM cases, although the numbers of patients
are small, there is a signicant benet of repeated local bcl-SR when used
in combination with systemic fotemustine therapy (biochemotherapy) with
no adverse effects.
161. SYSTEMIC TEMOZOLOMIDE COMBINED WITH
LOCOREGIONAL MITOXANTRONE IN TREATING
RECURRENT GLIOBLASTOMA PATIENTS
A. Silvani,
1
M. Eoli,
1
A. Salmaggi,
1
E. Lamperti,
1
A. Botturi,
1
G.
Broggi,
3
L. Bissola,
2
G. Finocchiaro,
2
and A. Boiardi
1
;
1
Neuro-oncology;
2
Unit of Experimental Neuro-oncology;
3
Neurosurgery, Istituto
Nazionale Neurologico Carlo Besta, Milan, Italy
The purpose of this study was to evaluate in recurrent glioblastoma
patients the feasibility and effectiveness of a combination treatment with
systemic temozolomide and locoregional mitoxantrone. The rst end point
of the study was the 6-month PFS; the secondary end points were response
rate and OS. Twenty-two recurrent GBM patients were enrolled for second
tumor debulking, with local positioning of a Rickam reservoir in order
to locally deliver chemotherapy into the tumor created resection cavity,
with the aim of controlling local tumor recurrence. We designed a protocol
using systemic temozolomide (200 mg/m
2
, days 15 every 28) in associa-
tion with mitoxantrone, delivered through the reservoir (4 mg, days 15
every 28) positioned into the area of tumor exeresis. After reoperation, a
residual tumor mass no larger than 2 cm was identied in 18/22 patients.
The patients were treated with monthly cycles of chemotherapy until evolu-
tion of the tumor but in no case for more than 10 cycles. Responses were
evaluated by MRI scans performed every two months and images assessed
according to McDonalds criteria. Response rates were as follows: no com-
plete responses [CRs], 5 partial responses [PRs], 13 cases of stable disease
[SD], and 4 cases of progressive disease [PD]. The median progression-
free survival [PFS] and survival time [ST] of the whole group of treated
patients was 7 and 11 months, respectively, and more than of the patients
survived over 18 months. During the study the patients compliance was
complete, and no dropouts occurred. Hematological toxicity was mild, and
after repeated local injections only minor neurological side effects occurred.
We conclude that although some bias in patient selection is not excluded in
this pilot study, results are interesting: The PFS was as long as survival of
recurrent GBM reported in the literature.
162. MANAGEMENT OF GLIOBLASTOMA MULTIFORME IN
ELDERLY PATIENTS: SHOULD WE BE AGEIST?
D. Rodrigues
1
, G. Hendry
1
, D. Scoones
2
, and P. Kane
1
;
1
Neurosurgery;
2
Neuropathology, James Cook University Hospital, Middlesbrough, UK
Past studies have suggested that aggressive treatment for glioblastoma
multiforme (GBM) in the elderly is not appropriate because of apparent
poor outcome. Global application of this view may deny some patients
benecial and state of the art treatment. Does this ageist view still hold
true? We conducted a retrospective audit of outcome in all elderly patients
(age 60 years) diagnosed with GBM in our institution between 2001 and
2003. The records were audited with respect to surgical treatment (biopsy,
resective surgery) and adjuvant treatment (radiotherapy, chemotherapy).
Outcome was analyzed on the basis of pre- and post-treatment Karnofsky
performance scale (KPS) and on survival. Statistical analysis performed
using Students t test and chi-squared test. Fifty eligible patients were iden-
tied from the neuropathology database (34 males, 16 females, mean age
of 68.9 6.3 years). For the purpose of analysis, patients receiving biopsy
alone, resective surgery alone, or biopsy plus radiotherapy were grouped
as a single group (Group I) and compared with outcome in patients receiv-
ing resective surgery plus radiotherapy chemotherapy (Group II). There
were 18 patients in Group I and 32 patients in Group II. The age in Group
I was 72.9 6.5 versus 66.5 5 in Group II (P 0.001). There was no
statistically signicant difference in the pre- and post-treatment KPS in the
2 groups. The mean survival in months in Group I was 3.61 2.9 and in
Group II was 9.7 5.5 (P 0.001). The mean time to last follow-up in
months was 6.6 5.2. Outcome in elderly patients with GBM in general
remains poor. However, elderly patients should not be treated as a homog-
enous group; as appropriately selected patients can have a good functional
and survival outcome. Further work is needed to determine the selection
criteria for treatment of elderly patients with GBM.
163. FACTORS PREDICTING SURVIVAL IN PATIENTS
WITH OLIGODENDROGLIAL TUMORS: EVALUATION
OF HISTOPATHOLOGY, MOLECULAR GENETICS,
NEUROIMAGING, AND THERAPY IN 210 PATIENTS
W. Shapiro, L. Ashby, J. Pueschel, A. Scheck, and S. Coons; Barrow
Neurological Institute, Neurology, Phoenix, Arizona, USA
Understanding outcome for oligodendroglial tumorsoligodendro-
glioma (O), oligoastrocytoma (OA), and their anaplastic counterparts
(AO, AOA)requires attention to patient-, tumor-, and treatment-related
variables. We present a large series of such patients. Two hundred ten
newly diagnosed patients were identied. Histopathological features and
proliferative activity (MIB-1) were determined, along with deletion of
chromosomes 1p and 19q (FISH). Preoperative MRIs were reviewed for
enhancement. Survival data and treatment information were obtained from
the clinical records. There were 139 low-grade tumors (95 O, 44 AO) and
71 high-grade tumors (31 AO, 40 AOA). Median age was 39 years (range,
1983) without age difference among the 4 subgroups. Patients aged 40
(MST 440 vs. 196 weeks; P 0.0002). Patients with low-grade tumors
lived longer than those with high-grade tumors (MST 446 vs. 138 weeks;
P 5% (P 0.001). Overall, patients with low-grade O or OA, and with
1p del lived longest. Patients whose tumors enhanced on MRI or had an
MIB-1 5% fared less well. Treatment did not inuence survival, but treat-
ment was not randomized prospectively. Multivariate analysis is currently
being performed.
164. HUMAN GLIAL TUMORS CONTAIN CELLS WITH
STEM CELL-LIKE PROPERTIES
C. Watts,
1,2
and S.L.R. Ball
2
;
1
Neurosurgery and
2
Cambridge Centre for
Brain Repair, Cambridge University, Cambridge, UK
Isolation of a population of multipotent stem-like cells that have sub-
sequently been shown to maintain glial tumors in vivo has recently been
reported. This suggests that despite the heterogeneity inherent in many
brain tumors a single cell type may be responsible for tumor growth and
maintenance. Understanding the relationship between brain tumors and
stem cells may help in the development of more effective therapies. Pro-
tocols developed for the culture of adult neural stem cells were applied
and modied for use with WHO IV glial tumor material from 5 patients.
Spheroid aggregates of tissue were derived. After serial passage in dened
propagation medium, clonal populations were transferred into differentia-
tion medium for 4 days, and phenotypic characterization was performed
by using standard immunohistochemical techniques. Multiple spheroid
colonies of slowly proliferating cells were identied from multiple tumor
samples. Serial passage of individual spheres yielded further expansion in
the number of spheres with prolonged cell cycling and slow proliferation
compared to typical tumor cells and normal mammalian adult neural stem
cells. Differentiation and characterization of single spheres identied cells
expressing neuronal lament B-tubulin consistent with a neuronal pheno-
type. Glial tumors appear to contain cells with characteristics of neural
stem cells that make them ideal candidates for malignant transformation.
Understanding stem cell signaling pathways and developing ways to manip-
ulate them will be critical in developing new treatments.
165. EPHA2 RECEPTOR AND ITS LIGAND, EPHRINA1, ARE
DIFFERENTIALLY EXPRESSED IN MALIGNANT GLIOMAS
AND NORMAL BRAIN
J. Wykosky, D. Gibo, and W. Debinski; Department of Neurosurgery,
Brain Tumor Center of Excellence, Wake Forest University School of
Medicine, Winston-Salem, North Carolina, USA
EphA2 is a member of the largest family of tyrosine kinase receptors,
the Eph receptors. These proteins play an important role in neural develop-
ment and are involved in mediating contact-dependent processes between
cells. EphA2 is expressed at low levels on the surface of adult epithelial cells,
where it interacts with its membrane-bound ligand, ephrinA1. The expres-
sion of EphA2 has been found elevated in a number of cancers, including
breast, colon, prostate, and ovarian carcinomas. In addition, the overex-
pression of this receptor has been linked to tumor neovascularization as
well as invasiveness. In our previous analysis using cDNA microarrays, we
found EphA2 to be one of the most uniformly overexpressed genes in glio-
blastoma multiforme (GBM). We were therefore interested in examining
the expression of the EphA2 receptor in GBM vs. that of normal brain.
The level of EphA2 was determined by Western blotting and immunohisto-
chemistry (IH) using both monoclonal and polyclonal EphA2 antibodies.
In Western blots, an immunoreactive band corresponding to the size of the
EphA2 receptor (130 kDa) was detected in all established human GBM cell
lines studied, including A-172, SNB-19, U-251 MG, U-87 MG, and U-373
MG, as well as in human GBM tissue lysates. Notably, EphA2 Western
blots of normal brain protein medleys obtained from Clontech or Chemicon
and lysates from the frontal lobe tissue of a normal human brain showed a
very faint EphA2 immunoreactive band at 130 kDa or no band at all. IH
performed on GBM cell lines revealed a honeycomb pattern of staining for
EphA2 that is characteristic of the expected membrane-localized expres-
sion. Furthermore, parafn-embedded and frozen sections of human GBM
display specic and abundant staining for EphA2 that was nearly absent
in normal brain. In addition, IH performed on a tissue microarray of 60
sections of various types and grades of brain malignancies demonstrated
intense EphA2 staining among the higher-grade (III and IV), more invasive
astrocytomas, but markedly less, if any, in the lower grade tumors. IH also
revealed that, unlike EphA2, ephrinA1 was present in parafn-embedded
sections of normal brain. Interestingly, ephrinA1 was localized specically
to vascular endothelial cells in normal brain, but present at a uniformly low
level throughout human GBM parafn-embedded specimens. Thus, EphA2
is specically overexpressed not only in malignancies of epithelial origin,
but also in malignant gliomas. Furthermore, EphA2 is expressed differen-
tially with respect to its ligand, ephrinA1, in both malignant gliomas and
normal brain. Overall, EphA2 represents a novel target for the development
of molecular therapeutics for the treatment of patients with high-grade glio-
mas. We are currently investigating the role of EphA2 and its interaction
with ephrinA1 as a useful means for diagnosis and therapeutic approaches
such as targeted drug delivery.
166. CHROMOSOME 10q LOSS IN MEDULLOBLASTOMA:
ASSOCIATION WITH THE LARGE CELL/ANAPLASTIC
VARIANT
C. Fuller,
1
J. Dalton,
1
and R. Gilbertson
2
;
1
Pathology;
2
Developmental
Neurobiology, St. Jude Childrens Research Hospital, Memphis,
Tennessee, USA
Medulloblastoma is by far the most common CNS embryonal tumor.
Although patient stratication is based primarily on clinical variables, dis-
tinctive histologic variants and molecular alterations have also been found
to correlate with prognosis. In particular, large cell/anaplastic (LC/A)
medulloblastoma characteristically presents with CSF dissemination and
follows a highly aggressive clinical course. Subsets of aggressive medullo-
blastomas have been found to harbor amplications of the c-myc oncogene,
many with concomitant LC/A morphology. Several studies have suggested
that deletions involving chromosome 10q may also play a role in medul-
loblastoma tumorigenesis, though no correlation has thus far been estab-
lished between these alterations and histologic subtype. Fluorescence in situ
hybridization (FISH) was performed on formalin-xed parafn-embedded
tissue sections from tissue microarrays containing 79 medulloblastomas
to determine c-myc and 10q status. Each case was designated as one of
the following histologic variants: classic, nodular/desmoplastic (N/D),
or large cell/anaplastic. Dual color hybridizations included the following
probe pairs: c-myc (8q24)/8p23.1, TNKS2 (10q23.32)/10p15.2, PTEN
(10q23.31)/DMBT1 (10q25.3-26.1). The cohort included 40 classic, 25
N/D, and 14 LC/A medulloblastomas. Overall, detectable amplications
of c-myc and losses involving 10q were present in 6 cases (8%) and 9 cases
(11%), respectively. In all cases with losses involving 10q, all three of the
targeted loci were deleted indicating a large region of chromosomal loss,
likely the entire long arm. Fifty percent of tumors with c-myc amplication
and 67% with 10q loss were of LC/A morphology. As a group, 43% of
LC/A medulloblastomas had 10q loss while 21% had amplication c-myc;
14% harbored both abnormalities. Only 5% of classic and 4% of N/D cases
had either alteration, and 2 of these cases had focal anaplasia. Our ndings
demonstrate that losses involving chromosome 10q are encountered in a
subset of medulloblastomas and involve a large region of that chromosomal
arm. Both c-myc amplication and 10q loss appear to correlate with the
LC/A morphology, the later alteration encountered twice as often as the
former in this series.
167. METHYLATION PROFILE OF PRIMARY CNS
LYMPHOMAS
L. Chi-Hang Chu, S. Grossman, and J. Herman; Johns Hopkins
University, Baltimore, Maryland, USA
Patients with systemic malignancies have been shown to have substan-
tial quantities of tumor-specic DNA in their plasma. Studies in systemic
malignancies are underway to determine if this can serve as a plasma marker
of tumor burden. We recently used methylation specic polymerase chain
reaction (MSP) to identify the methylation status of four gene promoters
(p16, p73, RARb, and MGMT) within resected brain tumor tissue and in
the plasma of patients with low- and high-grade gliomas. (Proc. Am. Soc.
Clin. Oncol. 2004). The presence of tumor-specic DNA in the plasma
was dened as identication of the same methylated promoter (MP) in the
primary brain tumor and the plasma. The gliomas contained methylation of
at least one promoter in 9 of the 10 (90%) patients studied, and 6 pts (67%)
had methylation of at least one of the same promoters in the plasma DNA.
A plasma marker would be of particular importance in patients with pri-
mary CNS lymphomas (PCNSL), where genuine improvements in outcomes
are being realized. In this study, we sought to characterize the methyla-
tion status of CNS lymphoma patients using a panel of 14 genes (p16
INK4a
,
p15
INK4b
, p14
ARF
, p73, Rb, hMLH1, GSTP1, MGMT, RARb, CRBP-1,
TIMP2, TIMP3, and DAPK). With IRB approval, we isolated DNA from
parafn-embedded samples from 31 CNS lymphoma patients and studied
the methylation patterns in the CpG islands of the genes using a nested
PCR approach. PCR product was diluted and amplied in the methyla-
tion-specic polymerase chain reaction (MS-PCR), which was performed
with primers specic for either methylated or modied unmethylated DNA
using positive and negative controls. The results showed a high degree of
aberrant methylation in PCNSL for DAPK (21/28 75%), p16
INK4a
(19/28
68%), CRBP-1 (16/25 64%), THBS-1 (17/28 61%), and p14
ARF

(13/24 54%); RARb (13/26 50%), MGMT (13/27 48%), TIMP2
(11/26 42%), TIMP3 (11/28 39%), and p15
INK4b
(12/31 39%).
This methylation prole provides an important rst step in developing a
quantitative tumor-specic plasma biomarker that could be used to monitor
tumor status in patients with PCNSL.
168. C-KIT, A POWERFUL TUMOR MARKER IN
GERMINOMA, IS MUTATED, AND TACE REGULATES C-KIT
SHEDDING IN GERMINOMAS
H. Nakamura,
1
K. Makino,
1
Y. Ushio,
2
and J. Kuratsu
1
;
1
Department
of Neurosurgery, Kumamoto University, Kumamoto;
2
Department of
Neurosurgery, Otemae Hospital, Osaka, Japan
The proto-oncogene c-kit encodes a transmembane tyrosine kinase
receptor for stem cell factor (SCF). C-kit is thought to have a crucial role
for tumor formation such as testicular seminomas, gastrointestinal stromal
tumors (GISTs), and intracranial germinomas. The soluble isoform of c-
kit (s-kit) is reportedly detectable in cerebral spinal uid of patients with
germinomas and germinomas with STGC. TACE (ADAM17), which is a
member of matrix-metalloproteinases, has been indicated to be a candi-
date for shedding of c-kit and generating a soluble isoform of c-kit (s-kit).
S-kit in CSF is now thought to be a powerful indicator for existence of
intracranial germinomas. In this study we performed immunohistochemi-
cal study on 25 human primary intracranial germinomas and germinomas
with STGC, staining the same sections for c-kit and placental alkaline
phosphatase (PLAP). Moreover, immunohistochemical staining for TACE
was performed in primary intracranial germinomas. The genomic DNA
was extracted from the germinomas and the mutation of c-kit gene was
examined. Immunohistochemical expression was graded by using a semi-
quantitative scoring system where 0%, 1 125%, 2 2650%,
3 5175%, and 4 76100%. Of the 25 cases, 7 (28%) were graded
3 and 18 (72%) 4 for c-kit; 8 (32%) were 3 or 4 for PLAP. All 3
cases negative for PLAP-staining were strongly positive for c-kit, and all
embryonal carcinomas, immature teratomas, and yolk sac tumors were
negative for c-kit staining. The positive reaction for TACE was conrmed
in germinomas and germinomas with STGC. However, it was negative in
other nongerminomatous germ cell tumors. C-kit gene mutation was found
in several cases of the patients with germinomas. We conclude that C-kit
has an important role for germinoma formation, and the mutation of c-kit
gene was conrmed in several cases. Both c-kit and s-kit may be powerful
tumor markers for germinomas with or without STGC. Moreover, TACE
is specically expressed on the surface of the tumor cells and generates s-kit
by shedding of c-kit in germinomas and germinoma STGC.
169. SURVIVAL OF ANAPLASTIC GLIOMAS CORRELATES
WITH GENETIC PROFILE
M. Sanson,
1
F. Laigle-Donadey,
1
J. Lejeune,
1
E. Criniere,
2
Y. Marie,
2

K. Mokhtari,
3
A. Carpentier,
1
K. Hoang-Xuan,
1
and J. Delattre
1
;
1
Neurology,
2
INSERM U495, and
3
Neuropathology, CHU Piti-
Based on WHO classication, anaplastic gliomas are divided into grade
III oligodendrogliomas (OIII), oligoastrocytomas (OAIII) and astrocyto-
mas (AIII). Known prognostic factors include age, performance status, and
genetic alterations such as 1p and 19q chromosome loss, 10q loss, and EGFR
amplication. Our aim was to investigate the impact of the most common
genetic alterations on survival, based on 739 anaplastic gliomas collected in
our database, including 445 males, 294 females (sex ratio 1.51) ranging
from 10 to 98 years (median 48 years). Genetic analysis included search
for loss of heterozygosity (LOH) on chromosome 1p and 19q, 9p, 10q,
EGFR and MDM2 amplication, P53 mutation and expression, PTEN
mutation: Partial or complete screening was available for 261 anaplastic
gliomas. Overall survival (OS) was 26.2 months and was correlated to age
(15.5 months for patients 48 years, vs. 47.9 months for patients 48
years; P 0.0001) but not to the histological subtype. Loss of 1p and 19q
(both linked P 10-11) were associated with OIII (P 0.001), 10q loss was
related to EGFR amplication (P 10-14), and to PTEN mutation (P
0.05). On univariate analysis, survival correlated with 1p loss (66.5 vs. 26.2
months, P 0.0001), 19q loss (65.3 vs. 26.2 months, P 0.005), 10q loss
(19.4 vs. 41.6 months, P 0.0001), PTEN mutation (11.1 vs. 36.1 months,
P 0.0004), EGFR amplication (16.6 vs. 37.1 months; P 0.0001),
MDM2 amplication (7.3 vs. 31.5 months, P 0.0025), P16/CDKN2A
deletion (18.9 vs. 33.1 months, P 0.013). The difference did not reach sig-
nicance for LOH 9p (P 0.064), p53 expression (P 0.11), or mutation
(P 0.9). LOH 10q and EGFR amplication were the only genetic altera-
tions correlated to age (P 0.0001), being more frequent in older patients
(51 vs. 43 years and 55.5 vs. 43 years, respectively). In conclusion, genetic
analysis of anaplastic gliomas provides useful prognostic information for
clinical practice and for stratifying clinical studies.
170. IMMUNOHISTOCHEMICAL EXPRESSION OF IMATINIB
TARGETS IN GLIOBLASTOMA: AN APPROPRIATE METHOD
TO SELECT PATIENTS FOR TARGETED THERAPY?
C. Haberler
1,2,3
;
1
Klin Institut of Neurologie,
2
Department of Internal
Medicine I,
3
Department of Neurosurgery, Medical University of Vienna,
Vienna, Austria
The tyrosine kinase (TK) inhibitor imatinib (Gleevec) selectively tar-
gets PDGFR-a, -, c-kit, c-abl, and arg and has proven successful in the
treatment of chronic myeloid leukemia. In recurrent glioblastoma, phase
2 therapy trials using imatinib have been initiated. As only a fraction of
patients seems to benet from imatinib therapy, and because of potential
side effects and high costs of imatinib therapy, selection of the right patients
is important. The goal of our study was to assess systematically immuno-
histochemical expression of the major TKs targeted by imatinib in glio-
blastoma, as expression of these factors could be used to select patients for
imatinib therapy. In a cohort of 101 glioblastoma patients, anti-PDGFR-a,
-, c-kit, c-abl, and arg protein immunohistochemistry was performed, and
expression of these proteins was assessed semiquantitatively. PDGFR-a and
arg expression in tumor cells was widespread in 1/101 cases, respectively.
Focal PDGFR-a, -, c-kit, c-abl, and arg immunolabeling was detected in
25/101, 19/101, 4/101, 7/101 and 31/101 cases, respectively. We show here
for the rst time in a large series of glioblastomas that PDGFR-a, -, c-kit,
c-abl, and arg expression is immunohistochemically detectable in a fraction
of cases. The value of anti-tyrosine kinase immunolabeling as predictive
factor for patient selection remains to be claried by comparative analysis
of tumor tissue of therapy-responders versus nonresponders.
171. GAMMA- CATENIN EXPRESSION CORRELATES WITH
GOOD PROGNOSIS IN MEDULLOBLASTOMAS
K. Misaki,
1
H. Fujisawa,
2
M. Hasegawa,
2
and J. Yamashita
2
; Toyama
Rosai Hospital, Neurosurgery, Uozu, Japan; Kanazawa University,
Neurosurgery, Kanazawa, Japan
In medulloblastoma, gene amplication and mRNA overexpression of
the c-myc gene are reported to be adverse prognostic indicators. However,
the frequency of mRNA overexpression (50%) cannot be explained by gene
amplication (4%) alone, and therefore, other mechanisms independent of
gene amplication may exist. Because c-myc is located downstream of the
Wnt signal pathway, we examined associated molecules in primary tumors
by immunohistochemical and cytogenetic analyses and have discussed their
clinical relevance. Twenty-four medulloblastomas were studied. Immuno-
histochemistry of c-myc, beta- and gamma-catenins, and cyclin D1 was
performed. Differential PCR was conducted for the gene amplication of
c-myc, N-myc, and cyclin D1. Mutations of beta- and gamma-catenins were
examined by PCR-SSCP analysis and direct DNA sequencing. Western blot
analysis was available in 5 cases. The clinical signicance of the results
was statistically analyzed by the Kaplan-Meier method. Cytoplasmic/mem-
branous staining of beta- and gamma-catenin was detected in 19 (79%)
and 9 (37%) cases, and nuclear expression of cyclin D1 and c-myc was
detected in 6 (25%) and 21 (83%) cases, respectively. The expression of
gamma-catenin in immunohistochemistry was conrmed by Western blot-
ting, and the expression levels were well correlated between the two. c-myc
and N-myc amplication was detected separately in two cases. Mutations
of beta- and gamma-catenins were not found. Statistically, patients without
CSF dissemination (Chang M0) showed signicantly better outcome than
those with dissemination (Chang M1~3) (P 0.0002), and only gamma-
catenin expression correlated with good prognosis (P 0.003) among the
molecules analyzed. Furthermore, gamma-catenin expression was also sig-
nicant in the M0 group (P 0.022). Although insignicant (P 0.057),
cyclin D1 expression showed a trend of adverse outcome, and all patients
with cyclin D1 expression expired. The expression of beta-catenin and c-
myc did not correlate with prognosis (P 0.31 and 0.53, respectively). The
immunohistochemistry of gamma-catenin is useful for further stratica-
tion or individualization in medulloblastoma treatment. It was also found
that cyclin D1 expression has the potential to be an adverse prognostic
indicator.
172. VH GENE ANALYSIS OF PRIMARY CNS LYMPHOMAS
H. Pels,
1
M. Montesinos-Rongen,
4
C. Schaller,
2
U. Schlegel,
1
I. Schmidt-
Wolf,
3
O. Wiestler,
1
and M. Deckert
2
;
1
Departments of Neurology,
2
Neurosurgery, and
3
Internal Medicine, University of Bonn, Bonn;
4
Department of Neuropathology, University of Cologne, Cologne;
5
German Cancer Research Center, Heidelberg; Germany
Primary CNS lymphomas are highly malignant non-Hodgkins lym-
phomas of B-cell origin associated with a poor prognosis. These neoplasms
show variable sensitivity to radio- and chemotherapy. A molecular basis
for these differences in treatment responses has not yet been established for
primary CNS lymphomas in a comprehensive series of patients. Here, we
performed PCR analyses of the immunoglobulin gene rearrangements of
18 PCNSL including nine patients who responded well to therapy and nine
patients who showed resistance to treatment. Variable gene segment dis-
tribution, mutation frequency of variable region genes, and clinical course
were analyzed. Our data suggest a tendency toward a higher mean muta-
tion frequency of variable region genes in patients responding to treatment
(17.2%) and a lower mutation frequency (11.8%) in patients exhibiting a
poor response to therapy, respectively. Furthermore, a restricted usage of the
VH4 gene family was observed in the majority of nonresponding patients.
To further validate the prognostic impact of these molecular parameters,
studies in a larger cohort of patients will be required.
173. PERINUCLEAR EXPRESSION OF LRIG PROTEINS
IS A GOOD PROGNOSTIC INDICATOR IN ASTROCYTIC
TUMORS
H. Hedman,
1
D. Guo,
1
J. Nilsson,
1
H. Haapasalo,
3
O. Raheem,
3

U. Andersson,
1
T. Bergenheim,
2
and R. Henriksson
1
;
1
Oncology and
2
Neurosurgery, Ume University Hospital, Ume, Sweden;
3
Tampere
University Hospital, Pathology, Tampere, Finland
The LRIG protein family has three members, LRIG13, which are
widely expressed integral membrane proteins. LRIG1 antagonizes the
activity of epidermal growth factor receptor (EGFR) family tyrosine kinase
receptors by enhancing receptor ubiquitylation and degradation. In this
study, we evaluated the mRNA expression level of LRIG13 in human
glioma cell lines and control-matched tumor tissues, characterized the sub-
cellular localization of a synthetic LRIG1-GFP fusion protein, and analyzed
the immunohistochemical staining patterns of LRIG13 in 404 astrocytic
tumors. LRIG13 mRNA was detected in all human glioma cell lines and
matched tumor samples. Ectopically expressed LRIG1-GFP localized to
nuclear, perinuclear, and cytoplasmic compartments in a cell line-specic
manner. Immunoreactivity of LRIG13 was seen in different patterns in
the astrocytic tumors. Perinuclear staining of LRIG13 inversely correlated
with WHO grade and survival of the patients. Positive LRIG3 perinuclear
and cytoplasmic staining correlated with a lower proliferation index.
LRIG3 perinuclear staining was in addition to tumor grade an independent
prognostic factor. Thus, expression of LRIG13 might be of importance in
the pathogenesis and prognosis of astrocytic tumors.
174. DEVELOPMENT AND APPLICATION RT-PCR SYSTEMS
TO DETERMINE HERV EXPRESSION IN ASTROCYTOMA
CELL LINES
P. Nelson, R. Smith, G. Conde, D. Roden, and J. Darling; University of
Wolverhampton, RIHS, Wolverhampton, UK
Human endogenous retroviruses (HERVs) belong to the family of trans-
posable elements that make up 8% of the human genome. Unlike exogenous
retrovirus (e.g., HIV and HTLV), HERVs are inherited in a Mendelian man-
ner. More than 22 families of HERVs have been identied over the past two
decades. Importantly, some HERVs have been found to possess large open
reading frames and produce viral like particles. More latterly, these viruses
have been linked with certain autoimmune diseases and cancers. Indeed,
HERVs may contribute toward carcinogenesis through retrotransposition,
promoter insertion, immunomodulation, disruption of normal HERV-
related functions, recombination, or by the production of fusion proteins.
Of importance, HERV-K, HERV-W, and HERV-H have the potential to
be transcriptionally active in the brain. We have developed robust RT-PCR
systems using primers/probes specic to HERV-K and HERV-W to assess
mRNA expression in conjunction with the house keeping gene, histidyl
tRNA synthetase. In employing a gel-documentation system, we are able to
provide semiquantitative levels of HERV expression in cell lines. Pilot data
shows markedly enhanced expression of HERV-K in the cell line U251-MG
(derived from a glioblastoma multiforme; WHO grade IV astrocytoma) as
compared to a control cell line SW480 (colon adenocarcinoma): RT-PCR
values; 1.0 and 0.42, respectively. This observation raises an intriguing
possibility that HERV-K expression may be elevated in malignant brain
tumors. In addition, this approach provides a useful approach to optimize
primers and probes prior to using real-time quantitative PCR.
175. MOLECULAR GENETICS AND RESPONSE TO PCV
CHEMOTHERAPY IN OLIGODENDROGLIAL NEOPLASMS:
A SINGLE CENTRE PROSPECTIVE STUDY
C. Walker
1
, B. Haylock
2
, D. Husband
2
, K. Joyce
1
, D. Fildes
1
,
M. Jenkinson
3
, T. Smith
4
, J. Broome
4
, D. du Plessis
3
, and P. Warnke
3
;
1
JK Douglas Research Laboratory, Clatterbridge Cancer Research Trust,
Bebington;
2
Clatterbridge Centre for Oncology, Bebington;
3
Department
of Neurological Science, University of Liverpool, Liverpool;
4
Walton
Centre for Neurology and Neurosurgery, Liverpool, UK
Recent research suggests that the -1p/-19q genotype is associated in
oligodendroglial neoplasms with prolonged survival and chemosensitivity.
However, the role of genetic analyses to guide patient management remains
controversial. In this prospective study, the impact of genotype on the
response of oligodendroglial neoplasms to procarbazine, lomustine, and
vincristine (PCV) chemotherapy was investigated in a routine clinical set-
ting. Seventy-six patients treated with PCV chemotherapy at a single center
between 2000 and 2003 were investigated. PCV was given as rst therapy
(50 cases) or at recurrence following radiotherapy (26 cases). Response
was assessed by using Macdonald criteria and T1-weighted MR or CT
images taken with contrast agents for enhancing tumors or T2-weighted
MR images for nonenhancing tumors. Allelic imbalance in 1p36, 19q13,
17p13, 10p12-15 and 10q22-26 and p53 mutation (exons 5-8) were inves-
tigated by using tumor samples enriched by laser capture microdissection.
The -1p/-19q genotype was found in 12/19 oligodendroglioma WHO grade
II, 10/23 oligoastrocytoma WHO grade II, 11/13 oligodendroglioma WHO
grade III, and 6/21 oligoastrocytoma WHO grade III. In enhancing tumors,
response was associated with the -1p/-19q genotype and inversely related
to loss of 17p13, chromosome 10 loss, and p53 mutation in the series as
a whole, or when Grade II and III or primary and recurrent cases were
analyzed separately. The -1p/-19q genotype was associated with longer
progression-free survival (PFS) and overall survival, while 17p13 loss, p53
mutation, and chromosome 10 loss indicated poor prognosis. Genotype
had greater impact in recurrent cases. Genetic factors remained indepen-
dent prognostic factors in multivariate analysis. In nonenhancing grade II
tumors, 4/6 with -1p/-19q responded to PCV as initial therapy compared
with 2/11 with intact 1p/19q. Nonenhancing cases with -1p/-19q showed a
trend toward longer PFS and signicantly greater time from rst to second
therapy. In the study overall, 26% of cases with intact 1p/19q responded
to PCV; 5 had p53 mutation, 3 had no detectable genetic alterations, and
1 had loss of 1p36 and chromosome 10. In addition to anaplastic tumors,
enhancing and nonenhancing grade II tumors with the -1p/-19q genotype
responded to PCV chemotherapy. This prospective study supports previous
retrospective observations that the -1p/-19q genotype is highly predictive
of chemosensitivity in oligodendroglial neoplasms, but some tumors with
intact 1p/19q also benet from PCV.
176. THERAPEUTIC IMPLICATIONS OF INTERFERON
REGULATORY FACTOR (IRF) -1 AND IRF-2 IN DIFFUSELY
INFILTRATING ASTROCYTOMAS (DIA): RESPONSE TO
INTERFERON (IFN) -BETA IN GLIOBLASTOMA CELLS AND
PROGNOSTIC VALUE FOR DIA
A. Yoshino, Y. Katayama, T. Yokoyama, T. Watanabe, A. Ogino, T. Ota,
C. Komine, and T. Fukushima; Neurological Surgery, Nihon University
School of Medicine, Tokyo, Japan
The precise mechanisms governing the direct effect of IFN-b, including
apoptosis induction, are not yet fully understood. To gain a better insight
into these mechanisms, we investigated the signaling pathways focusing
particularly on interferon regulatory factor 1 (IRF-1) and IRF-2 in glio-
blastoma cell lines. Furthermore, we attempted to determine whether or not
IRF-1 and IRF-2 act as additional prognostic indicators in diffusely inl-
trating astrocytomas (DIA). We rst assessed the cytotoxic effects of IFN-b
based on a cell growth study and modied MTT assay, and then quantied
the apoptosis using a sandwich enzyme immunoassay following IFN-b treat-
ment in the cell lines, U-87MG, T98G, and A-172. Subsequently, we carried
out an analysis of apoptosis-related molecules as evaluated by densitometric
analysis of Western blots, focusing on IRF-1 and IRF-2, and 2 major initia-
tor caspases, caspase-8 and caspase-9. Furthermore, we assessed the expres-
sion of type I IFN receptor, IRF-1, and IRF-2 using immunohistochemical
techniques in 63 DIA (15 of WHO grade II, 18 of grade III, and 30 of
grade IV), and analyzed their impact on prognosis. An increase in apoptosis
was apparent after 48 h of IFN-b treatment (1 10
4
IU/ml) in T98G but
not in U-87MG or A-172. IFN-b treatment for 6 h signicantly enhanced
the expression of IRF-1 in all 3 cell lines. However, an enhanced expres-
sion of IRF-2 was observed only in the not-most-sensitive, non-apoptosis-
induced U-87MG and A-172. While minimal processing of caspase-8 was
noted in the 3 cell lines throughout the experiment, caspase-9 activation
was observed in the apoptosis-detected T98G after 48 h of treatment, as
indicated by a 1.33-fold increase (P 0.037). On the other hand, the IRF-1
LI and IRF-1/IRF-2 LI ratios were greater in low-grade DAI and were nega-
tively correlated with the histopathological grade in DIA (P 0.017 and
P 0.001, respectively). Furthermore, the IRF-1/IRF-2 LI ratio was nega-
tively correlated with the MIB-1 LI in DIA (P 0.004) and represented an
independent and most powerful determinant of overall survival compared
to other conventional prognostic factors (P 0.018). However, the relation
was not statistically signicant when only patients with high-grade DIA
were assessed. Our ndings suggest that upregulation of IRF-1 and IRF-2
might be an important determinant of susceptibility to IFN-b-mediated
cytotoxicity including apoptosis. Furthermore, the IRF-1/IRF-2 LI ratio
may reect the proliferative state of DIA and constitute an important prog-
nostic marker in DIA. Thus, IRF-1 and IRF-2 could represent one of the
therapeutic target sites for the regulation of cell growth in DIA.
177. PROGNOSTIC SIGNIFICANCE OF
O6-METHYLGUANINE-DNA METHYLTRANSFERASE
(MGMT) PROTEIN EXPRESSION IN ADULT ANAPLASTIC
GLIOMAS
M. Brell,
1
A. Tortosa,
2
J.J. Acebes,
1
J.M. Gil,
3
T. Ribalta,
6
E. Verger,
7

S. Vill,
4
N. Violas,
8
I. Ferrer,
5
and F. Graus
9
;
1
Neurosurgery and
2
Nursery Department, Bellvitge Hospital,
3
Oncology and
4
Radiation
Therapy Department, Institut Catal dOncologia,
5
Bellvitge Hospital,
Institut of Neuropathology, Universitat Barcelona, LHospitalet de
Llobregat, Barcelona;
6
Pathology,
7
Radiation Therapy,
8
Oncology, and
9
Neurology Departments, Hospital Clinic of Barcelona; Spain
O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair
protein that removes mutagenic and cytotoxic adducts from the O6 position
of guanine. MGMT activity in glioma cells could be related to resistance
to alkylating agents. The aim of this study was to analyze MGMT protein
expression in anaplastic gliomas and to establish their relationship among
tumor characteristics, patient survival, and MGMT methylation status of
the promoter. Ninety-three patients with anaplastic glioma (WHO grade
III) were analyzed for MGMT protein expression by immunohistochemis-
try on parafn-embedded sections. MGMT promoter methylation prole
was analyzed in 40 specimens by methylation specic PCR. We evaluate
the prognostic implication of patients epidemiological data (including age
and gender), MRI scan characteristics (diffuse, ring, and no enhancement),
extent of resection, histopathology, postoperative KPS, proliferation index
(Ki-67 expression), and MGMT immunohistochemical expression. Overall
survival was calculated according to the Kaplan-Meier method and com-
pared with the log-rank test. A multivariate analysis was carried out accord-
ing to proportional-hazard analysis (Coxs model). Sixty (64.5%) patients
were men, and the median age of the patients was 48 years. Median overall
survival was 18 months. Fifty-one tumors (54.8%) showed nuclear staining
of MGMT. No correlation between MGMT expression and promoter meth-
ylation was observed. Coxs regression analysis revealed that postoperative
KPS 80, proliferative index 4.8%, absence of contrast enhancement on
MRI and gross total resection were independent predictors of longer overall
survival. There was a trend toward longer overall survival for those patients
with negative MGMT immunohistochemistry (RR 1.66, P 0.066). In a
subset analysis of patients who received chemotherapy (n 72), absence of
MGMT expression was independently associated with better survival (RR
2.12, P 0.027). Unlike previous investigators, we did not nd a cor-
relation between MGMT promoter methylation and survival. However, we
observed a correlation between MGMT protein expression and survival in
those patients who received chemotherapy. This previously reported feature
will need to be validated in future studies.
178. ESTHESIONEUROBLASTOMA: A STUDY OF
PROGNOSIS
V.G. Khurana, C. Giannini, M. Piccirilli, D.M. Bernold, W.R. Bamlet,
R. Foote, J. Buckner, R.L. McClelland, and M.J. Link; Pathology, Mayo
Clinic Rochester, Rochester, Minnesota, USA
Esthesioneuroblastoma is a rare tumor, for which experience at individ-
ual institutions has been limited. At present, there is no consensus regard-
ing its systematic treatment. In order to determine prognostic factors for
outcome in the management of this tumor, we retrospectively studied the
records of 78 patients treated for esthesioneuroblastoma at the Mayo Clinic
between 1976 and 2003. Histologic slides from original biopsy/surgery
were reviewed in 60 (of 78) patients, and Hyams pathologic grade (12,
low; 34, high) was determined. Statistical analysis included Kaplan-Meier
overall and disease-free survival and Cox proportional hazards modeling,
uni- and multivariate, to assess the strength of association of the following
variables with survival: age and gender, Hyams grade, modied Kadish
stage (A to D), extent of surgery, and adjuvant treatment. There were 35
females (45%). The average patient age at diagnosis was 51 years and aver-
age follow-up was 6.4 years (Hyams grade, n low, 31; high, 29; modied
Kadish stage, n A, 4; B,15; C,42; D,15). Fifty-four patients underwent
gross total resection, 16 subtotal, and 8 biopsy only, while 51 patients
received radiotherapy and 31 chemotherapy. The 5-year survival (68%
overall) was: 84% for low and 50% for high grade tumors (P 0.004);
88% for stages A and B, 72% for stage C, and 34% for stage D patients
(P 0.05). Controlling for age and extent of resection, stage D exhibited
signicantly higher mortality than either stage A B (P 0.0005) or stage
C (P 0.006), while high-grade tumors had signicantly higher mortality
than low grade (P 0.007). In total, 53 patients were at risk for recurrent
disease, and 32 (60%) of these went on to develop recurrence: 9% at 1 year,
36% at 5 years, and 77% at 10 years. Our data suggest that both modied
Kadish stage and Hyams grade at the time of diagnosis have a signicant
impact on prognosis.
179. GENETIC ANALYSIS OF MENINGIOMAS REVEALS
CHROMOSOME-SPECIFIC CORRELATIONS WITH GRADE
AND RECURRENCE
E.J.M. Speel,
1
S. Claessen,
1
J. Stavast,
2
E. Meulemans,
2
G. Blaauw,
3

J. Teepen,
4
R. Janzer,
5
and A. Twijnstra
3
;
1
University of Maastricht,
Molecular Cell Biology, Maastricht, The Netherlands;
2
Pathology
and
3
Neurology, Academic Hospital Maastricht, Maastricht, The
Netherlands;
4
St. Elisabeth Hospital, Neurology, Tilburg, The
Netherlands;
5
University of Lausanne, Neuro-Pathology, Lausanne,
Switzerland
Meningiomas are common central nervous system neoplasms that
exhibit a remarkably diverse histopathology and biological behavior. Apart
from the involvement of the NF2 gene, the molecular pathology of menin-
gioma development and progression is poorly understood. We examined
25 benign (WHO grade I; MI), 10 atypical (WHO grade II; MII) and 6
anaplastic, malignant (WHO grade III; MIII) meningiomas, including 14
tumors of 5 patients showing recurrence (n 3) and progression (n 2),
for DNA copy number gains and losses by comparative genomic hybridiza-
tion (CGH) and for chromosome 1, 7, and 22 alterations by uorescence in
situ hybridization (FISH) using centromere- and NF2 genespecic probes.
In addition, 44 tumors of 12 patients showing malignant progression were
studied by microsatellite analysis using 7 markers for loci on chromosomes
1p and 22q. Results were statistically correlated with available clinical data.
CGH analysis identied (1) genomic alterations in all but 6 MI tumors; (2) a
marked accumulation of the average number of alterations per tumor with
increasing WHO grade, i.e., 5.2 for MI, 10.0 for MII, and 11.8 for MIII (P
0.001; M1 vs. MII III); (3) besides 22q losses most frequently 17pq and
20q gains (36%40%) in MI, losses of 1p, 14q (both 50%), 6q (40%), and
gains of 17q (70%), 20q (60%), 17p (50%), and 11q (40%) in MII, and a
further increase of most of these alterations (1p losses and 17q gains; 83%),
as well as amplications on 5p15, 5q35, 8q24, and 20q13 in MIII tumors;
(4) a signicant association of 1p and 6q losses and 15q gains with higher
WHO grade (MI vs. MII/MIII; P 0.05); and (5) a strong correlation of
tumor recurrence with 1p and 10p losses (P 0.057 and 0.006, respec-
tively) and 1q gains (P 0.032), as well as with an increase in total number
of alterations and losses per tumor, i.e., 5.7 vs. 11.5 (P 0.007) and 2.4 vs.
6.0 (P 0.003), respectively. FISH analysis in these tumors showed chro-
mosome 1 and 7 copy numbers indicating a diploid DNA content, while
1 copy of the NF2 locus was lost in 18/23 (78%) MI, 8/9 (89%) MII and
5/5 (100%) MIII tumors, predominantly together with a chromosome 22
centromere. Microsatellite analysis revealed LOH for at least 1 marker at
22q in 8/10 and at 1p in 7/11 additional patient cases showing progression,
which was already detected in the primary tumor. Our study indicates that
meningiomas are mainly diploid tumors showing high frequencies of chro-
mosome 22q loss including the NF2 gene as well as 17pq and 20q gains
already early in tumorigenesis. Tumor recurrence proved to be associated
with 1p and 10p losses and 15q gains, as did 1p and 6q losses and 15q gains
with tumor progression. The identied chromosomal regions may lead to
the discovery of novel genes that play a role in meningioma tumorigenesis
and may help to predict their biological behavior. This study was supported
by the Royal Netherlands Academy of Sciences (KNAW), Van Leersum
Foundation, and the Foundation Neurosurgery Heerlen.
180. PROSPECTIVE ANALYSIS OF MATRIX
METALLOPROTEINASE-9 (MMP-9) IN THE SERUM OF
PATIENTS WITH HIGH-GRADE GLIOMAS
A. Hormigo,
1
B. Gu,
2
E. Riedel,
3
J. Rao,
5
M. Fleisher,
2
K. Panageas,
3

L. DeAngelis,
1
and E. Holland
4
;
1
Neurology,
2
Clinical Laboratories,
3
Biostatistics, and
4
Neurosurgical Service of the Department of Surgery,
Memorial Sloan-Kettering Cancer Center, New York, New York;
5
Biomedical and Therapeutics Sciences, University of Illinois College of
Medicine, Peoria, Illinois, USA
The objective of the study is to determine if matrix metalloprotein-
ase-9 (MMP-9) can be used as a serum marker in patients with malignant
gliomas. Our previous work has shown that the level of YKL-40 protein
can predict survival of patients with high-grade gliomas. However, the
YKL-40 does not signicantly correlate with response to therapy. We have
now identied MMP-9 as a potential marker for response to therapy in
gliomas. MMPs are proteases that degrade extracellular matrix proteins,
and MMP-9 is expressed by human gliomas and associated with tumor
inltration and angiogenesis. Serum MMP-9 levels were determined by
ELISA assay prospectively and correlated with magnetic resonance imaging
(MRI) scans in a prospective longitudinal study. The tumor response was
determined by standard criteria: complete response (CR), partial response
(PR), stable disease (SD) and progression of disease (POD) for each MRI.
We analyzed serum samples from 57 patients with anaplastic gliomas (AG)
and 48 patients with glioblastoma multiforme (GBM) (range, 112 samples;
median of 4 and 3 per patient, respectively). For AG the mean value of
MMP-9 was 323 ng/ml for patients who had a radiographic CR, 196 ng/ml
for PR, 372 ng/ml for SD, and 519 ng/ml for POD. For GBM, the mean
value of MMP-9 was 190 ng/ml for CR, 356 ng/ml for PR, 425 ng/ml
for SD, and 526 ng/ml for POD. To determine if MMP-9 levels predict
the response categories, we used a cumulative logit model that corrected
for multiple measurements per patient. This analysis showed a signicant
difference of MMP-9 values per response type for AG (P 0.002) and a
marginally signicant difference for GBM (P 0.08). Similar to what is
seen with YKL40, the MMP-9 levels transiently increase immediately fol-
lowing surgery reaching the maximum level at 48 h after tumor resection
(mean 1174 ng/ml and 1070 ng/ml, respectively) and decreased over the
next two weeks. Therefore, reliable levels must be obtained at least two
weeks post-surgery. In conclusion, serum levels of MMP-9 correlate with
response type at least for AG. Ongoing longitudinal studies are designed
to (1) assess correlation with survival and (2) determine whether MMP-9
can complement YKL-40 as a reliable serum marker for disease status in
patients with high-grade gliomas and be used to guide clinical decisions
during a patients disease course.
181. CLINICAL SIGNIFICANCE OF EGFR AMPLIFICATION
AND THE EGFRVIII TRANSCRIPT IN CONVENTIONALLY
TREATED ASTROCYTIC GLIOMAS
L.M. Backlund,
1
L. Liu,
2
B.R. Nilsson,
1
D. Grander,
1
K. Ichimura,
2

and V.P. Collins
2
;
1
Oncology-Pathology, Karolinska Institute,
Stockholm, Sweden;
2
Department of Pathology, Division of Molecular
Histopathology, Addenbrookes Hospital, University of Cambridge,
Cambridge, UK
The aim of this study was to evaluate the clinical value of assessing
amplication and the common 5 rearrangement of the EGFR gene (EGFR-
vIII) in 223 astrocytic tumors by correlating the data with patient survival.
Previous studies have evaluated amplication alone and provided contradic-
tory results. Amplication was analyzed by densitometry of Southern blots
or quantitative PCR, and the EGFR transcripts were examined by RT-PCR
and sequenced to identify aberrations. In addition, RNase protection assay
was carried out on a subgroup of the tumors to conrm the PCR results. We
found no signicant association between EGFR amplication or rearrange-
ment and survival in a series of 160 glioblastoma patients. There was no
association between EGFR status and age in this patient group. We noted
a tendency toward decreased survival in the 42 patients with anaplastic
astrocytomas with any EGFR abnormality. This was most marked for the
EGFRvIII rearrangement (P 0.061). The latter anaplastic astrocytoma
patients were signicantly older than those without 5 rearrangements
(P 0.020). No EGFR abnormalities were identied in the astrocytoma
patients. We conclude from this large set of astrocytic tumors that neither
EGFR amplication nor the presence of the EGFRvIII transcript predict
patient outcome in conventionally treated glioblastomas. In anaplastic
astrocytomas, however, although uncommon, EGFR aberrations are asso-
ciated with shorter survival.
182. MGMT PROMOTER HYPERMETHYLATION IS MORE
FREQUENT IN SECONDARY GLIOBLASTOMAS AND IS
INDEPENDENT FROM OTHER PROGNOSTIC FACTORS
M. Eoli, L. Bissola, F. Menghi, B. Pollo, A. Silvani, L. Valletta, G.
Broggi, A. Boiardi, and G. Finocchiaro; Neurological Institute C. Besta,
Clinical Neuro-oncology, Milan, Italy
O
6
-methylguanine-DNA methyltransferase (MGMT) is a DNA repair
protein that specically removes mutagenic, carcinogenic, and cytotoxic
O
6
-alkylguanine DNA adducts induced by alkylating agents like nitrosou-
reas. Repair of cytotoxic DNA damage by MGMT is a potentially impor-
tant factor of resistance to alkylating agents, commonly used in the treat-
ment of glioblastoma multiforme (GBM). Using methylation-specic PCR
(MSP) we investigated the inactivation of the DNA-repair gene MGMT
by promoter hypermethylation in 67 GBMs obtained from patients sub-
sequently treated by conventional radiotherapy and CDDP BCNU. We
observed that the MGMT gene was methylated in 24 patients (36%). This
nding was associated with prolonged overall survival (24 vs. 14 months;
log-rank P 0.0002) and with a longer progression-free survival (PFS)
(11 vs. 8 months; log-rank P 0.003). Among these 67 GBMs, secondary
GBMs had prolonged overall survival (26 vs. 14 months; log-rank P 0.01)
than de novo tumors, whereas other prognostic factors were not statistically
associated with ST or PFS. Moreover, the frequency of MGMT methyla-
tion was higher in secondary than in primary GBMs (78% vs. 22%, P
0.01), but was not associated with age, KPS, and RTOG class risk. Other
genetic markers (LOH on chromosome 19q and chromosome 17p, EGFR
amplication and p53 mutations) are under study to assess their inuence
on the treatment response and overall survival of patients with GBM. These
ndings indicate the relevance of epigenetic and genetic factors in the prog-
nosis of glioblastomas.
183. HYPOXIA INDUCIBLE FACTOR-1 ALPHA EXPRESSION
CORRERATES WITH HISTOLOGICAL GRADING IN HUMAN
GLIOMAS
M. Saino, K. Sakurada, Y. Sonoda, S. Sato, and T. Kayama; Department
of Neurosurgery, Yamagata University School of Medicine, Yamagata,
Japan
Hypoxia inducible factor-1 (HIF-1) is considered to play an important
role in the adaptation of cells to hypoxia and angiogenesis via regulation
of vascular endothelial growth factor (VEGF) in various tumors. Astrocy-
tomas, especially glioblastoma and anaplastic astrocytoma, are known to
be hypervascular tumor. We investigated the relationship between HIF-1
alpha expression and several factors such as VEGF expression, Ki-67 label-
ing index, and histological grading in human gliomas. Expression of HIF-1
alpha, VEGF, and Ki-67 antigen were investigated by immunohistochem-
istry in 35 specimens of supratentorial astrocytomas (15 glioblastomas,
11 anaplastic astrocytomas, and 9 low-grade astrocytomas). Histological
grading was determined according to WHO criteria. Strong and moderate
expression of HIF-1 alpha was observed in 6 (40%) of 15 glioblastomas,
in 7 (63.7%) of 11 anaplastic astrocytomas, and 2 (22.2%) of 9 low-grade
astrocytomas. The case did not show HIF-1 alpha expression was observed
in 4 (26.7%) of 15 glioblastomas, 3 (27.3%) of 11 anaplastic astrocytomas,
and 5 (45.5%) of 9 low-grade astrocytomas. The cases with strong and
moderate expression of HIF-1 alpha tend to show strong VEGF expression.
The mean Ki-67 labeling index was 20.4% in the strong and moderate HIF-
1 alpha expression group and 8.5% in the no HIF-1 alpha expression group.
There was correlation of HIF-1 alpha expression with VEGF expression,
Ki-67 labeling index, and histological grading. Malignant astrocytomas
widely expressed HIF-1 alpha, which contributes to angiogenesis via VEGF.
Furthermore, HIF-1 alpha would correlate with tumor cell proliferation.
These results suggest that neovascularization due to upregulation of HIF-1
prevents hypoxic damage and permit tumor cell progress.
184. MICROGLIA IN GEMISTOCYTIC ASTROCYTOMAS
F. Geranmayeh,
1
B.W. Scheithauer,
2
and M.B. Graeber
1
;
1
University
Department of Neuropathology, Imperial College London and
Hammersmith Hospitals Trust, London, UK;
2
Department of Laboratory
Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, USA
Gemistocytic astrocytomas behave more aggressively than other
diffusely growing astrocytic tumors. They are characterized by a high
mutation rate of the p53 gene and cytological as well as immunological
abnormalities including frequent perivascular mononuclear infiltrates.
Microglia are resident brain macrophage precursor cells that form a net-
work of immune competent cells within the normal central nervous system.
They are of increasing interest in the context of glioma growth. We selected
23 tumor samples from among 201 samples obtained from patients with
gemistocytic astrocytomas operated on at the Mayo Clinic between 1985
and 1998. These tumors have been previously analyzed for p53 mutations,
p53 protein, and proliferative activity (Neurosurgery 48, 187 2001). Immu-
nolabeling for three established microglial markers, CR3/43, Ki-M1P, and
iba1 was carried out on adjacent tissue sections. A high number of micro-
glial cells were detected in gemistocytic astrocytomas. More microglia were
present when the fraction of gemistocytes was high. Varying numbers of
gemistocytic tumor cells expressed MHC class II molecules. Importantly,
the more class II immunoreactive gemistocytes were present, the fewer
class II positive microglial cells could be detected. Our ndings support the
view that gemistocytic astrocytomas contain unusually high numbers of
microglial cells. The nding of aberrant MHC class II expression by gem-
istocytic tumor cells correlating with a loss of immune competent, MHC
class IIexpressing microglia sheds new light on the immunology of these
tumors. We suggest that the ndings reported here may be related to the
especially poor prognosis of gemistocytic astrocytomas. It is well known
that the proliferative potential of neoplastic gemistocytes is very low, and
it has remained an intriguing question as to why these tumors are so suc-
cessful biologically. The fact that aberrant expression of MHC class II
molecules by nonprofessional antigen presenters may induce T cell anergy
could provide one explanation.
185. PROGNOSTIC VALUE OF MGMT METHYLATION, TP53
MUTATION, AND MDM2, EGFR, OR CDK4 AMPLIFICATION
IN MALIGNANT GLIOMA PATIENTS TREATED WITH
ADJUVANT TEMOZOLOMIDE CHEMOTHERAPY
C. Luyken,
1
S. Loeser,
2
B. Blaschke,
2
S. Khler,
1
A. Kreuzmann,
1
S.
Seghrouchni,
1
G. Reifenberger,
2
and M. Sabel
1
;
1
Clinic of Neurosurgery;
2
Department of Neuropathology, Heinrich-Heine-University, Dsseldorf,
Germany
The response of malignant gliomas to chemotherapy with alkylating
drugs such as temozolomide (T) varies from patient to patient. The present
study investigates the signicance of tumor-associated genetic aberrations
in the TP53, MDM2, EGFR, and CDK4 genes, as well as the methyla-
tion of the MGMT promoter for response to T and overall survival (OS)
of patients with malignant gliomas. Fifty-one patients with malignant
gliomas (anaplastic WHO grade III [AG], n 14, WHO grade IV [GBM],
n 37) were treated with T as rst-line chemotherapy after tumor resection
and radiation therapy. From each patient, clinical data were systematically
recorded, including age at diagnosis, gender, tumor location, Karnofsky
performance score (KPS), extent of resection, tumor volume (determined
before and after resection, before chemotherapy, and every 3 months), OS,
and response to treatment. The tumor tissue was investigated for muta-
tions in the TP53 gene (exons 59); amplication of the EGFR, CDK4 and
MDM2 genes; and hypermethylation of the MGMT promoter. Molecular
ndings were correlated to OS and response to T. Fifty-nine percent of
the patients were males, and median age at surgery was 54.3 years. The
mean preoperative KPS was 80%. OS was 277.4 weeks (AG) and 87 weeks
(GBM). A median number of 8 cycles of T was administered. Grade 3 or
4 toxicity occurred in 6 patients. T was terminated because of tumor pro-
gression in 31 patients (4 AG, 29%; 27 GBM, 73%). Thirty-three percent
of the tumors demonstrated EGFR amplication (17% AG, 38% GBM),
while MDM2 amplication was detected in 7% (8% AG, 6% GBM), CDK4
amplication in 16 % (8% AG, 18% GBM), and TP53 mutation in 20%
(12,5% AG, 22% GBM). MGMT hypermethylation could be demonstrated
by PCR in 54% of the cases (82% AG, 46% GBM). Statistical analyses
revealed that MGMT hypermethylation was signicantly associated with
longer OS (P 0.0013, log-rank test) and better response to T (P 0.034,
log-rank test). Neither EGFR, MDM2, and CDK4 amplication nor TP53
mutation was signicantly correlated to OS or response to T. In line with
recent publications from other groups, our study clearly indicates MGMT
hypermethylation as a clinically important molecular marker that is sig-
nicantly associated with longer overall survival and better response to
temozolomide treatment in patients with malignant gliomas. In contrast,
genetic alterations in the TP53, MDM2, EGFR, and CDK4 genes appear to
be less important as prognostic factors. This study was supported by grants
(70-3088-Sa I) from Deutsche Krebshilfe.
186. SECRETORY MENINGIOMAS AS MORPHOLOGICALLY
DISTINCT MENINGIOMA ENTITY
H.D. Mennel,
1
T. Riegel,
2
W. Tirakotai,
2
and I. Celik
3
;
1
Department of
Neuropathology,
2
Clinic of Neurosurgery, and
3
Institute of Theoretical
Surgery, Philipps University, Marburg, Germany
The unique features, both concerning clinical and pathological aspects,
of secretory meningioma have not been completely understood so far. We
present a hitherto not noticed pathological nding that could shed new
light upon this rare entity. Fourteen cases of secretory meningiomas could
be identied out of more than 1300 meningiomas from our institutions
during the last 25 years. Tumors were retrospectively analyzed regarding
their clinical and pathological features with special emphasis upon mecha-
nisms that might explain the clinically observed common edema. The main
morphological nding was the occurrence of a signicantly higher propor-
tion of mast cells in secretary meningiomas compared to other unselected
meningioma subtypes: mean 2.43 (range, 0.0205.60) versus 0.27 (range,
0.002.00; P 0.0001, Mann-Whitney-U-test). These mast cells can be
visualized and counted with the CD-117 immunoreaction, an antibody
directed against the product of the c-kit oncogene. Mast cells were identi-
ed in close connection to vessel wall (pericytic) proliferations. Electron
microscopy of such proliferated vessels revealed increasing vacuolization
in the peripheral layers of proliferated vessels, constantly adjacent to small
edematous areas, in which the mast cells often were freely oating. Yet, we
could not establish a positive correlation between microscopic mast cell
density and macroscopic edema as found by CT scans in respective cases.
Furthermore, the pseudopsammoma bodies that characterize those tumors
are surrounded by cells that react vividly with cytokeratins. Cytokeratin
subgroups such as CK 7, CK 8, and CK 5/6 are equally found at random and
in combinations; only CK 20 was regularly absent from those cells. Further
attempts to characterize mast cells and secretory products by a panel of
tissue hormones (serotonin and others) failed to give clues as to the action
of mast cells and to the pathogenesis of pseudopasmmomam bodies (secre-
tory products) within these tumors. Pericytic vessel proliferation, edema
formation of secretory meningiomas, and the newly described occurrence
of mast cells may well be correlated. The further analysis of these features
and of their connection to the formation of pseudopsammoma bodies is
difcult because these tumors are as rare, only approximately 1% of all
meningiomas.
187. DOES MR SPECTROSCOPY PREDICT SURVIVAL IN
GLIOMAS?
J. Crossman,
1
D. Birchall,
2
P. Mitchell,
1
and P. Crawford
1
;
1
Departments
of Neurosurgery and
2
Neuroradiology, Newcastle General Hospital,
Newcastle Upon Tyne, UK
The WHO grading of a glioma is based on observed histopathologi-
cal features. While the grade predicts the behavior of the tumor, it is only
approximately related to length of patient survival following diagnosis. MR
spectroscopy of gliomas is well correlated with the histopathological grade.
However, the use of MR spectroscopy data to predict duration of survival
following diagnosis is not widespread. We have investigated the use of a Cox
proportional hazards model using MR spectroscopy data obtained from a
prospective series of histopathologically proven gliomas to predict duration
of survival following diagnosis. We have compared the accuracy of this
model with four other models based on WHO grade, tumor histopathology,
and the MRI characteristics of the tumor. MR spectroscopy data was pro-
spectively acquired with a Philips 1.5T scanner using multivoxel chemical
shift spectroscopy. Standard post-acquisition processing of the MR spec-
troscopy data was performed, and the following ratios were calculated:
(i) choline/creatine, (ii) choline/naa, (iii) choline/creatine
normal
, (iv) lactate/
creatine
normal
, (v) choline/choline
normal
, (vi) choline
normal
/creatine
normal
,
(vii) choline
normal
/naa. Five Cox proportional hazards models were con-
structed, (i) WHO grade, (ii) histopathological features of the tumor, (iii)
MR spectroscopy, (iv) MRI characteristics of the tumor, and (v) combined
MRI/MR spectroscopy. Each model also included covariates for age at
diagnosis and information on whether surgical debulking and/or further
oncological treatment occurred. Because some of the patients remained
alive at the time of analysis, the Kaplan-Meier algorithm was used to cal-
culate the survival function. The survival predicted by each model was
compared to the actual survival at three months, one year, and two years.
Thirty patients (19 males) were studied. The age range at diagnosis was
26 to 79 years (mean, 57 years). Eighteen patients were WHO grade IV, 8
patients WHO grade III, and 4 patients WHO grade II. Actual survival at
3 months was 87%, at one year 50%, and at two years 43%. The histo-
pathology and MR spectroscopy models most closely predicted the actual
survival (MR spectroscopy: 3 months 94%, one year 59%, two years 45%;
histopathology: 3 months 92%, one year 58%, two years 48%). All of the
models overpredicted survival, particularly in the rst year. This investiga-
tion demonstrates that MR spectroscopy is a useful tool to help estimate
survival following diagnosis of a glioma and suggests its use in conjunction
with other data known to be of prognostic value.
188. 1p/19q STATUS IN RECURRENT OLIGODENDROGLIAL
TUMORS: WHAT DOES IT TELL US?
I. Lavon,
1
B. Zelikovitsh,
1
D. Fuchs,
1
Y. Fellig,
2
D. Soffer,
2
and T. Siegal
1
;
1
Leslie and Michael Gafn Center for Neuro-Oncology and
2
Department
of Pathology, Hadassah Hebrew University Hospital, Jerusalem, Israel
Oligodendrogliomas frequently show allelic loss of chromosomal arms
1p and 19q. These deletions are associated with an improved response to
chemotherapy and longer overall survival when compared to tumors with
no such allelic loss. The assessment of the genetic status of 1p/19q is rou-
tinely performed by analyzing the primary neoplasm, while the implication
of the re-evaluation of the recurrent tumor is unclear. Our objective was
to evaluate the status of 1p and 19q in both the primary and the recurrent
oligodendroglial tumors. The status of 1p/19q was evaluated from paired
tumor-blood DNA samples by using PCR-based microsatellite analysis. Ten
tumors of 5 patients were evaluated and included the primary and recurrent
neoplasm. For 3 patients, initial diagnosis was oligodendroglioma (WHO
II, 2 pts; WHO III, 1 pt). Two of these tumors had codeletions at 1p and
19q chromosomes, and one had no loss. At recurrence, the two low-grade
tumors transformed to a higher grade and were now diagnosed as anaplas-
tic oligodendroglioma (WHO III). The repeat evaluation of 1p/19q status
showed again, a combined allelic loss. The anaplastic tumor with no initial
loss remained 1p/19q. Two other patients had an initial diagnosis of
an oligoastrocytoma, one low grade and the other anaplastic (WHO III).
The low-grade tumor had codeletions at 1p/19q, and the anaplastic tumor
had an intact 1p with a deletion at 19q. At recurrence, both tumors were
rediagnosed by a stereotactic biopsy, obtained from a part of the tumors
that demonstrated radiographic changes but were located at sites of the
residual tumors that were not resected on initial surgery. The pathology of
the low-grade tumor has not changed, but no deletions were found in the
recurrent neoplasm. The anaplastic tumor transformed to WHO IV grade,
and this recurrent tumor presented no deletions. Our ndings may indi-
cate that pure oligodendrogliomas are of monoclonal origin, while mixed
oligoastrocytomas probably contain a biclonal population. It is possible
that different parts of the heterogenous tumors represent different clonal
expansion. Alternatively, it can not be excluded that over time and fol-
lowing treatment, the more resistant clones that do not manifest deletions
overtake and become dominant.
189. ANAPLASTIC ASTROCYTOMA PATIENTS WITH
GLIOBLASTOMA-LIKE TUMOR GENOTYPE HAVE A POOR
OUTCOME
L.M. Backlund,
1
B.R. Nilsson,
1
L. Liu
2
, K. Ichimura,
2
and V.P. Collins
2
;
1
Oncology-Pathology, Karolinska Institute, Stockholm, Sweden;
2
Department of Pathology, Division of Molecular Histopathology,
Addenbrookes Hospital, University of Cambridge, Cambridge, UK
Anaplastic astrocytoma (AA, WHO grade III) is, second to glioblas-
toma, the most common and most malignant type of adult CNS tumor.
Since survival for patients with AA varies markedly and there are no known
useful prognostic or therapy response indicators, the primary purpose of
this study was to examine whether the knowledge of genetic abnormalities
in AA had any value in this regard. The survival data on 37 carefully sam-
pled AA was correlated with the results of a detailed analysis of the status
of 9 genes involved in the development of astrocytic tumors. These included
3 genes coding for proteins in the p53 pathway (i.e., TP53, p14ARF, and
MDM2), 4 in the Rb1 pathway (i.e. CDKN2A, CDKN2B, RB1 and CDK4)
and PTEN and EGFR. We found that abnormalities in at least one of the
four genes (CDKN2A, CDKN2B, RB1, CDK4) coding for components of
the Rb1 pathway were associated with shorter survival (P 0.009). This
nding was consistent in multivariate analysis, including adjustment for
age (P 0.013). The ndings suggest that analysis of the genes coding for
Rb1 pathway components provides additional prognostic information in
AA patients.
190. PRIMITVE NEUROECTODERMAL TUMOR ARISING
WITHIN LOW- GRADE ASTROCYTOMA. A REPORT OF
THREE CASES
D. Pal,
1
G. Hall,
1
A. Chakrabarty,
2
P. Roberts,
3
and P. Chumas
1
;
1
Neurosurgery and
2
Neuropathology, Leeds General Inrmary;
3
Cytogenetics, St. Jamess University Hospital, Leeds, UK
We present three cases of primitive neuroectodermal tumor (PNET)
arising in low-grade astrocytomas treated with radiotherapy. One case was
identied at clinical presentation, two from a review of our histopathology
database. All three patients had histologically proven low-grade astrocy-
tomas and had received radiotherapy following biopsy. Two patients had
partial resection for recurrence, one at ve years post-surgery and the other
at ten years with histological conrmation of low-grade astrocytoma. At
subsequent recurrence at the same site, nine and 29 years following origi-
nal presentation and nine years later for the third patient, further tumor
debulking was performed. Histology now showed PNET (WHO grade 4).
Two patients died within one year of nal surgery. The third patient is
still alive, but with evidence of clinical progression. Cytogenetics showed
a complex karyotype with multiple chromosome abnormalities in all three
patients; recurrent abnormalities included deletions of 1q32, 2p13, 13q14,
and 19q13. PNET arising in a previous low-grade astrocytoma has not
been described previously. Two reports in the literature describe PNET
arising at a distant site following radiotherapy for low-grade astrocytoma.
Whether these cases represent transformation of low-grade astrocytoma
into PNET or PNET arising de novo in the previous radiotherapy eld will
be discussed.
191. MOLECULAR CHARACTERIZATION OF PDGF
SUBFAMILY OF RECEPTOR TYROSINE KINASES PATHWAY
IN GLIOSARCOMAS
R.M. Reis,
1
A. Martins,
1
S.A. Ribeiro,
1
D. Basto,
1
A. Longatto-Filho,
1

F. Schmitt,
1
and J.M. Lopes
2
;
1
Life and Health Sciences Research
Institute (ICVS), Health Sciences School, University of Minho, Braga;
2
Institute of Molecular Pathology and Immunology of University of
Porto, Porto, Portugal
Deregulation of PDGF subfamily of receptor tyrosine kinases, such
as PDGFR-a and c-Kit, and anomalous activation of RAS-RAF-MAPK
intracellular signaling pathway have a crucial role in the development and
progression of several neoplasms. GISTs are characterized by frequent c-Kit
oncogenic mutations and less commonly by PDGFR-a mutations. Activat-
ing mutations of B-RAF, a member of RAF kinase family, are frequently
found in several tumors, mainly melanomas. The evaluation of this pathway
is of great importance, owing to the fact that signal transduction mediated
by c-Kit and PDGF receptors can be efciently blocked by specic tyro-
sine kinase inhibitors such as Imatinib (Gleevec, Novartis). Gliosarcoma
is a rare and poorly characterized malignant brain tumor that exhibits a
biphasic tissue pattern with areas of glial and mesenchymal differentia-
tion. Molecular studies showed that gliosarcomas display a genetic prole
similar to glioblastomas and support the concept of monoclonal origin of
both components. The aim of this study was to characterize the molecular
alterations of PDGF subfamily of receptor tyrosine kinases pathway in glio-
sarcomas. Immunohistochemistry for PDGF-A, PDGFR-a and c-Kit were
analyzed in both components of six gliosarcomas. Activating mutations
in PDGFR-a (exons 12 and 18) and c-Kit (exons 9, 11, 13, and 17) genes
were studied by PCR followed by direct sequencing. B-RAF gene (exons 11
and 15) was screened by using PCR-SSCP followed by direct sequencing.
Expression of PDGF-A was found in all cases: three with similar moderate
to intense immunoexpression in both glial and mesenchymal components,
two with predominant intense expression in the glial component, and one
case with predominant moderate mesenchymal expression. Co-expression
of PDGFR-a was observed in three cases, mainly in glial component. c-Kit
immunopositivity was observed in both components of one case. The muta-
tional analysis of PDGFR-a showed the presence of a 2440-49_50insA
intronic insertion in two cases, and a 2472CT silent mutation in two
cases. No mutations were detected in c-Kit and B-RAF genes. PDGF-A
was overexpressed in gliosarcomas. PDGFR-a was overexpressed in 50% of
cases, predominantly in the glial component supporting an autocrine loop
gliosarcomas; overexpression of c-Kit was not a frequent event. Activating
mutations of PDGFR-a and c-Kit were not found, suggesting that other
mechanisms are responsible for overexpression of these receptor tyrosine
kinases in gliosarcomas.
192. EXPRESSION OF CD15 ON NEOPLASTIC CELLS OF
INTRINSIC BRAIN TUMORS: A MODULATORY ROLE IN
METASTASIS?
G. Pilkington,
1
A.J. Bolteus,
2
J. Rees,
3
R. Folkerth,
4
K. Asano,
5
and
P. Forsyth
6
;
1
School of Pharmacy & Biomedical Sciences, University of
Portsmouth, UK;
2
Department of Neurosurgery, Yale University School
of Medicine, New Haven, Connecticut, USA;
3
Institute of Neurology,
University College, London, UK;
4
Harvard Medical School, Boston,
Massachusetts, USA;
5
Department of Neurosurgery, Hirosaki University,
Hirosaki-shi, Japan;
6
Tom Baker Cancer Centre, University of Calgary,
Alberta, Canada
CD15, the adhesive sialyl LewisX oligosaccharide, acts as the cellular
ligand for E-, L- and P- selectins as well as binding homophilically. It is
widely distributed in epithelial cells and granulocytes. Within the develop-
ing normal brain neurones, astrocytes, oligodendrocytes, and Bergmann
glia have also been reported to express this epitope. CD15 is also strongly
expressed in many highly metastatic somatic cancers and is thought to
mediate tumor cell adhesion to vascular endothelium prior to extravasa-
tion, enabling entry to, and colonization of, new sites for metastatic spread.
We have previously shown that CD15 is expressed by experimental rat and
mouse gliomas but that human glioma cells in vitro are invariably negative
(Martin et al, Anticancer Res. 15, 1159, 1995). The ability of such glioma
cells to adhere to non-brain vascular endothelial cells was also seen to be
poor. In the present study we have examined archival histological sections
from 19 cases of intrinsic brain tumor (including astrocytoma, anaplas-
tic astrocytoma, oligodendroglioma, and glioblastoma multiforme) from
which extraneural metastatic spread was apparent. In each of these cases,
taken from ve different hospitals, sections of the original tumor plus the
secondary deposit (in chest wall, lymph nodes, scalp, bone marrow and
lung) were examined by immunohistochemical staining using the DAKO
anti-CD15 antibody (MO733). While CD15 expression was seen in inl-
trating hematogenous cells, and discounted, expression on neoplastic cells
within both primary and secondary sites was seen. Corresponding primary
site control cases for the various histological types of tumor that had
not been seen to metastasize showed CD15 positivity for neoplastic cells
in only one case (glioblastoma multiforme). We conclude and postulate
that expression of CD15 in primary brain tumors may indicate a possible
propensity to spread outside the nervous system by virtue of facilitating
adhesion of neoplastic glia to non-brain vascular endothelium. The Saman-
tha Dickson Research Trust is gratefully acknowledged for their support
of this research.
193. YKL- 40 EXPRESSION IS ASSOCIATED WITH
ACTIVATED PI3K AND MAP-KINASE PATHWAYS IN
GLIOBLASTOMA: IMPLICATIONS ON AGE AND SURVIVAL
C. Pelloski, A. Mahajan, S. Woo, and K. Aldape; Radiation Oncology,
The University of Texas M.D. Anderson Cancer Center, Houston, Texas,
USA
We have previously shown that YKL-40 is overexpressed in glioblas-
toma (GBM) and its overexpression is associated with poorer response to
radiation and reduced survival time. YKL-40 has been shown to activate
the AKT and MAP kinase (MAPK) pathways in vitro. Therefore in this
study, we test for an association of YKL-40 with activated intermediates of
these pathways in GBM tumor specimens. The interaction between age and
molecular marker status, with respect to overall survival (OS), is analyzed
as well. The sample set comprised 294 patients with newly diagnosed GBM
who underwent resection from 1994 to 2003. Immunohistochemical stain-
ing was performed on patient tissue for YKL-40, activated (phospho-) spe-
cies of the AKT pathway (p-AKT, p-mTOR, and p-70S6K) and p-MAPK.
Staining was scored as positive or negative. Clinical factors associated with
decreased OS included older age and lower KPS (all P 0.003, Kaplan-
Meier). Expression of YKL-40, p-mTOR, p-70S6K and p-MAPK were
highly correlated with each other (all P 0.009, Spearman). There was a
trend in correlation between YKL-40 and p-AKT. Expression of YKL-40,
p-AKT, and p-MAPK correlated with advanced age (all P 0.01). YKL-40,
p-mTOR, p-70S6K, and p-MAPK expression levels were associated with
decreased overall survival within the entire patient set (all P 0.02,
Kaplan-Meier). However, nearly all of the survival variation explained by
the markers was observed within the young patient population. Specically,
among patients younger than the median age (59 years), univariate analyses
showed YKL-40, p-mTOR, p-70S6K, and p-MAP to be prognostic factors
(all P 0.01). Multivariate analysis (Cox regression) revealed that lower
KPS (HR 3.8, P 0.03) and YKL-40 positivity (HR 1.8, P 0.03) were
independent prognostic factors after adjusting for extent of surgery, and
p-mTOR, p-70S6K, and p-MAPK staining scores. None of these factors
were signicant in univariate analysis within the older patient group. The
in vitro observation that YKL-40 activates the AKT and MAPK pathways
is supported by the demonstrated correlation with these activated species in
patient tumor specimens. While these markers are in general more prevalent
in older patients, the survival impact of these markers on younger patients is
far greater than on older patients. These ndings may explain the survival
differences between older and younger patients with GBM. The presence
of these markers in patients younger than the median age for GBM (5560)
should be considered in risk stratication.
194. INVESTIGATION OF THE EGFR- GENE
AMPLIFICATION STATUS DETERMINED BY FISH AS A
PREDICTIVE OR PROGNOSTIC MARKER FOR PATIENTS
WITH RECURRENT HIGH- GRADE GLIOMA TREATED
WITH TEMOZOLOMIDE
B. Neyns,
1
J. Sadones,
1
B. Van Mierlo,
1
C. Chaskis,
2
A. Michotte,
3

J. Menten,
6
E. Joosens,
7
T. Strauven,
5
G. Storme,
4
and J. De Greve
1
;
1
Medische Oncologie,
2
Neurochirurgie,
3
Anatomopathologie/
Neurologie, and
4
Radiotherapie, Academisch Ziekenhuis Vrije
Universiteit Brussels, Brussels;
5
Neurologie, AZ St-Augustinus,
Antwerpen;
6
Radiation-Oncology, Universitair Ziekenhuis Gasthuisberg
Katholieke Universiteit Leuven, Leuven;
7
AZ-Middelheim, Medische
Oncologie, Antwerpen; Belgium
The epidermal growth factor receptor (EGFR) gene is frequently ampli-
ed and mutated in high-grade glioma (HGG). Immunohistochemical stain-
ing for the EGFRvIII mutant is a negative prognostic factor for patients with
anaplastic glioma (Buckner et al., J. Clin. Oncol. 22 (14S), 1508, 2004). We
investigated the prognostic and predictive value of EGFR-gene amplication
determined with uorescence in situ hybridization (FISH) in HGG treated
with temozolomide (Temodal, TMZ) at recurrence following surgery and
radiotherapy. Clinical data and tumor material were collected from a retro-
spective cohort of patients treated at 4 Belgian hospitals. All patients were
treated with TMZ at recurrence. In addition to conventional diagnostic
histopathology, HGG were characterized by FISH for the amplication
status of the EGFR-gene (Vysis, LSI EGFR/CEP 7 Dual Color Probe). Our
patient population consisted of 36 men and 22 women (N 58) with a
median age of 58 years (range, 1680 years). At the initiation of TMZ
treatment, tumors consisted of 14 AA/AOA and 44 GBM. Eighteen tumors
were found to have an amplication of the EGFR gene (4/14 AA/AOA,
14/44 GBM). No signicant association was found between EGFR-gene
amplication and sex, age (at diagnosis or at initiation of TMZ), glioma
localization, type of surgery (resection versus biopsy), histology (at diag-
nosis or at initiation of TMZ), the interval between diagnosis of HGG and
the initiation of TMZ, or response to TMZ (objective response or disease
control). We found no signicant correlation between EGFR-gene ampli-
cation and time to progression or overall survival following the initiation of
TMZ treatment (Kaplan-Meier survival statistics, log-rank test, P 0.3).
In a subgroup analysis of the 17 patients with an AA/AOA or secondary
GBM, EGFR-gene amplication was associated with an inferior overall
survival (log-rank test, P .04). We could not demonstrate a predictive
or prognostic value of EGFR-gene amplication in recurrent HGG treated
with TMZ. Our observations support the concept that EGFR-amplica-
tion is a negative prognostic factor for OS in patients with an AA/AOA or
secondary GBM.
195. LOW SERUM S100 LEVELS IN PATIENTS WITH
NEWLY DIAGNOSED LUNG CANCER CORRELATE WITH
AN ABSENCE OF BRAIN METASTASES ON MRI
M.A. Vogelbaum, P. Mazzone, T. Masaryk, T. Mekhail, V. Fazio, S.
McCartney, N. Marchi, A. Kanner, and D. Janigro; Cleveland Clinic
Foundation, Brain Tumor Institute, Cleveland, Ohio, USA
Early detection of metastatic brain tumors in patients with a known
systemic cancer may provide these patients with less invasive treatment
options. We have previously observed that disruption of the blood-brain-
barrier (BBB), which occurs in the setting of metastatic brain tumors, is
accompanied by a release of the astrocytic protein S100 into the serum
where it can be detected by use of a simple, relatively low cost assay. We
hypothesized that measurement of S100 in the serum may serve as a screen-
ing test for cerebral metastases in individuals with lung cancer. Individuals
with newly diagnosed non-small cell lung cancer were enrolled in the study
if there were no symptoms or signs of neurologic dysfunction suggestive
of the presence of metastatic brain tumors. Each participant underwent
MRI of the brain and had serum drawn with measurement of the S100
level. The level of S100 was compared to the results from MRI imaging.
Thirty-eight individuals agreed to participate in the trial. Seven of the 38
had evidence of metastatic disease on MRI of the brain, and 22 had normal
MRI scans. The remaining 9 patients had no brain metastases but had T2
changes consistent with chronic microvascular disease. The S100 level was
lower in those with normal MRIs than those with chronic microvascular
disease or metastatic disease (0.07, 0.49, and 0.28 g/liter respectively; P
0.002 for patients with normal MRIs compared to those with chronic
microvascular disease, and P 0.03 for patients with normal MRIs com-
pared to those with metastatic disease). Utilizing a cutoff of 0.12 mg/L
the sensitivity of S100 for cerebral metastatic disease was 100%, speci-
city was 13%, negative predictive value was 1, and positive predictive
value (PPV) was 0.47. If patients with chronic microvascular disease were
excluded, the specicity was 86% and the PPV was 0.88. Low serum levels
of S100 ( 0.12 g/liter) accurately identied lung cancer patients who
do not have metastatic brain tumors. However, an elevated serum S100
in this group of patients does not specically indicate the presence of brain
metastases. Nevertheless, the results of this study suggest that assessment of
serum S100 can be used to narrow the population of lung cancer patients
who may benet from periodic surveillance imaging.
196. LOSS OF HETEROZYGOSITY (LOH) OF 1p IN
OLIGODENDROGLIAL TUMORS IS RELATED TO REDUCED
EXPRESSION OF NUCLEAR FACTOR KAPPA B (NFKB)
I. Lavon,
1
D. Fuchs
1,
, Y. Fellig,
2
Z.B.B. Zelikovitsh,
1
T. Siegal
1
;
1
Leslie
and Michael Gafn Center for Neuro-Oncology;
2
Department of
Pathology, Hadassah Hebrew University Hospital, Jerusalem, Israel
In oligodendrogliomas, LOH on chromosome 1p is associated with
tumor chemosensitivity. The molecular mechanism for this association is
unknown. Nuclear factor kappa B (NFkB) is a transcription factor found
in the cytoplasm as hetero- or homodimer that exerts its effects through
binding to nuclear DNA and activating transcription of target genes. While
the ultimate gene targets of NFkB are diverse, its activation can block apop-
tosis in numerous tumor cell lines. Moreover, NFkB inactivation is associ-
ated with increased susceptibility of tumor cells (including glioma cells) to
antineoplastic therapy, probably secondary to facilitated apoptosis. Many
tumors exhibit constitutive activity of NFkB, which possibly contributes
an antiapoptotic effect associated with chemoresistance. We postulated
that the chemosensitivity of oligodendroglioma may be related to reduced
NFkB activation. Our objective was to evaluate NFkB activation in oligo-
dendrogliomas and its relationship to 1p status. We evaluated 41 oligo-
dendroglial tumors (WHO II, 26; WHO III, 15) for their LOH status by
PCR. Activated NFkB which undergoes nuclear translocation was assessed
by immunohistochemistry (IHC) of the p65 subunit. The level of NFkB
activation was dened as high (50%100% nuclear staining), intermediate
(10%50%) or low (10% nuclear staining). All tumors (100%) with an
intact 1p (n 17) had high activation of NFkB as opposed to 43% (10/23)
of tumors with 1p LOH. NFkB level of activation differed signicantly (P
0.02) between tumors with or without 1p LOH. This difference was more
pronounced in the WHO II group, where high activation was found in all 11
tumors with intact 1p but in only 30% of those with 1p loss (P 0.0004).
We suggest that low NFkB activation plays a role in the chemosensitivity
of oligodendrogliomas with LOH at 1p. Recent publications demonstrated
that TNFa has a central role in NFkB activation and in protection against
apoptosis. The deleted region on 1p (1p36) contains some of the tumor
necrosis factor receptor super family (TNFRSF) such as TNFR2 that was
shown to mediate NFkB activation. Further investigations are needed in
order to conclude whether a deletion of TNFRSF at 1p site can induce che-
mosensitivity as a result of reduced NFkB activation.
197. DETERMINATION AND PROGNOSTIC SIGNIFICANCE
OF MITOTIC INDEX IN GRADE II AND III INFILTRATING
ASTROCYTOMAS USING THE MITOSIS MARKER
PHOSPHO-HISTONE H3
K. Aldape,
1
G.C.H. Fuller,
1
L. Huang,
1
C. Giannini,
2
and P. Burger
3
;
1
The University of Texas M.D. Anderson Cancer Center, Houston,
Texas;
2
Mayo Clinic, Rochester;
3
Johns Hopkins School of Medicine,
Baltimore, Maryland, USA
Histologic classication of inltrating astrocytomas into malignancy
grades II or III depends primarily on the presence of mitotic activity. The
accuracy in determining mitotic activity can be affected by the amount of
available tumor for study as well as cellularity of the tumor. Methods to bet-
ter quantify mitotic activity may improve the ability to stratify patients into
prognostic groups. Phospho-histone H3 (pHH3) is specically upregulated
in mitosis, and anti-pHH3 immunohistochemistry facilitates the detection
of mitotic gures in parafn sections. Cases of newly diagnosed supraten-
torial astrocytomas in adults (18 years) were identied in the pathology
records. Archival parafn material (total 123) was obtained for cases
of grade II astrocytoma (A, n 20) or grade III (anaplastic) astrocytoma
(AA, n 103). A representative section from each case was stained for
pHH3. Mitoses were identied by positive staining as well as morphologic
conrmation of features of mitosis. The area of tumor with the highest
mitotic activity on low power examination was selected for mitotic index
determination. The mitotic index was determined by counting the number
of positively stained mitoses per 1000 tumor cells. The mitotic index ranged
from 0 to 20 mitoses per 1000 tumor cells, with a median of 2. In univariate
analysis, higher mitotic index was a signicant predictor of poorer survival,
and this remained signicant (P 0.01) in multivariate Cox analysis after
adjustment for age and tumor grade. After condensing the mitotic index
data into 2 groups with low and high mitotic activity, Kaplan-Meier analy-
sis indicated that cases with a mitotic index of 0, 1, or 2 (n 66) had a
signicantly improved (P 0.04) median survival (434 weeks) compared
with cases with an index of 3 or above (n 57, 177 weeks). We conclude
that pHH3 staining provides a means to rapidly identify mitotic gures in
order to obtain a true mitotic index. Determination of mitotic index offers
theoretical advantages in the quantication of mitotic activity compared
with current practices. Higher mitotic index is a signicant independent
predictor of survival in grade II and grade III astrocytomas. An index of
2 mitoses or less per 1000 cells in the most mitotically active area appears
to dene a group of tumors with survival time typical of grade II tumors,
while an index of 3 or greater portends a prognosis typical of grade III
anaplastic astrocytoma.
198. NESTIN, CXCL12/SDF1, PDGFR-BETA AND VEGF
EXPRESSION IN ENDOTHELIAL PROLIFERATION ON 80
GLIOMAS: CLINICOPATHOLOGICAL CORRELATIONS AND
SUGGESTIONS FOR ANGIOGENESIS
B. Pollo,
1
E. Maderna,
1
C. Calatozzolo,
2
and A. Salmaggi
2
;
1
Neuropathology and
2
Neuro-oncology, Istituto Nazionale Neurologico
C. Besta, Milan, Italy
Nestin is a class VI intermediate lament (IF), highly expressed in
embryonic progenitor cells and is a marker for multipotential neuroepithe-
lial stem cells, involved in early stages of lineage commitment, in prolifera-
tion and in differentiation. Nestin is detected in neuroepithelial tumors and
in endothelial cells in active proliferation. The VEGF expression upregu-
lates CXCR4 on endothelial cells, binding the proangiogenic chemokine
SDF1/CXCL12 (Stromal Derived Factor). CXCL12 have a role on angio-
genesis and chemotaxis of endothelial cells throughout the activation of
his receptor. PDGF has a role in glial tumorigenesis and modulates angio-
genesis, and its receptor PDGFR-beta may be overexpressed in gliomas on
angiogenic endothelial cells. To investigate the amount and the meaning
of endothelial proliferating cellexpressed nestin related to the presence
of VEGF, SDF1/CXCL12, and PDGFR-beta in gliomas, we performed an
immunohistochemical study, with their specic antibodies, on 80 patients
with gliomas of different phenotype and malignancy grade. The patients
were categorized by disease: 20 glioblastomas, 12 anaplastic astrocyto-
mas, 12 anaplastic oligoastrocytomas, 4 anaplastic oligodendrogliomas,
10 astrocytomas, 12 oligoastrocytomas, and 10 oligodendrogliomas. More-
over, all the cases were immunostained for GFAP, vimentin, Ki67/MIB1,
and CD34. Nestin was variably expressed in neoplastic cells and in endothe-
lial proliferations, suggesting a correlation to malignancy grade and clinical
outcome. The immunodetection of nestin and CXCL12 in some endothelial
cells in low-grade gliomas with shorter time to tumor progression suggests
a role in early angiogenesis. A correlation was found also to expression of
VEGF and PDGFR-beta. These results suggest that some cells expressing
nestin, a marker of neural progenitor cells, stimulated by growth factors
and chemokines, may be involved in angiogenesis as proliferating endothe-
lial cells. Our data, especially in low-grade gliomas, might provide useful
information related to angiogenesis and might add prognostic information
in patients with variable life expectancy.
199. LOSS OF HETEROZYGOZITY ON CHROMOSOME
1p 19q IS A MAJOR FAVORABLE PROGNOSTIC FACTOR
FOR PROGRESSION-FREE SURVIVAL IN LOW- GRADE
GLIOMAS
F. Laigle-Donadey,
1
J. Lejeune,
1
E. Criniere,
2
Y. Marie,
2
M. Kujas,
3
L.
Capelle,
4
M. Sanson,
1
K. Hoang-Xuan,
1
and J. Delattre
1
;
1
Neurologie,
2
INSERM U495,
3
Neuropathologie, and
4
Neurochirurgie, CHU Piti
The morphologic classication of low-grade gliomas remains contro-
versial and insufcient. To investigate whether commonly described genetic
alterations have prognostic signicance, search for loss of heterozygosity
(LOH) on chromosome 1p, 9p, 10q, 19q, EGFR amplication, and p53
expression was performed in a series of 131 low-grade gliomas. The prole
of molecular alterations was then correlated with clinical parameters and
course of the disease, mainly progression-free survival (PFS). There was a
correlation between an oligodendroglioma phenotype and LOH 1p 19q
(P = 1.4. 10-8), and between an astrocytoma phenotype and P53 expression
(P 3. 10-7). On multivariate analysis, only LOH on chromosome 1p was
associated with increased PFS (RR 0.52), while histology and clinical
parameters were not signicant. This study indicates that LOH 1p is the
single most important prognostic factor for PFS in low-grade gliomas.
200. A GENETIC POLYMORPHISM OF THE CHEK2
GENE AS A PROGNOSTIC FACTOR IN GLIOBLASTOMA
MULTIFORME
M. Simon,
1
R. Fimmers,
2
M. Ludwig,
3
P. Sssmann,
1
J. Oldenburg,
4

and J. Schramm
1
;
1
Department of Neurosurgery,
2
Institute of Medical
Statistics and Informatics, and
3
Institute of Clinical Biochemistry,
University Hospital Bonn, Bonn;
4
Institute for Transfusion Medicine,
University Hospital Frankfurt/Main, Frankfurt/Main, Germany
CHEK2 gene germline mutations have been identied in some fami-
lies with Li-Fraumeni syndrome. Li-Fraumeni patients suffer from various
malignancies including glioblastoma multiforme (GBM). The CHEK2 gene
is located on chromosome 22q12.2. Up to 30% of sporadic GBMs may har-
bor LOH (loss of heterozygosity) for markers from chromosome 22q. The
CHEK2 gene product is a negative cell cycle regulator and important effec-
tor of the DNA damage control pathways. We tested the hypothesis that a
genetic polymorphism of the CHEK2 gene might modify the incidence and
the clinical course of sporadic human GBM. DNA was isolated from periph-
eral blood of 213 patients with primary GBMs and 192 control subjects.
Samples were genotyped with respect to a biallelic SNP (single nucleotide
polymorphism), CHEK2 IVS1, -711 A/T, using PCR and restriction digests.
For all patients the following clinical parameters were recorded: age, sex,
histology (GBM NOS, giant cell GBM, gliosarcoma), localization, degree
of resection (biopsy, partial, subtotal and gross total resection), postopera-
tive radiotherapy, postoperative chemotherapy, postoperative Karnofsky
performance score. No association between the CHEK2 SNP and GBM
incidence was found. Interestingly, the CHEK2 IVS1, -711 genotype cor-
related signicantly with survival (P 0.034, log-rank test), most clearly
among patients receiving chemotherapy (n 28, P 0.0008). Highly sig-
nicant clinical prognostic factors were age, degree of resection (biopsy
vs. cytoreductive surgery), postoperative radiation and chemotherapy, and
postoperative Karnofsky score (all P 0.0001, log-rank test). This study
conrms the important role of well-known clinical prognostic factors such
as age, Karnofsky performance index, and postoperative radiotherapy. Our
data support the use of cytoreductive surgery and chemotherapy for GBMs.
Most interestingly, this investigation suggests a genetic polymorphism as a
prognostic marker for GBMs. Since CHEK2 is involved in the detection
and repair of DNA damage (e.g. after chemotherapy), possible explana-
tions include differential chemosensitivity mediated by different CHEK2
IVS1, -711 alleles.
201. MORPHOLOGICAL AND MOLECULAR
HETEROGENEITY WITHIN TUMORS WITH
OLIGODENDROGLIAL CHARACTERISTICS
M.C.M. Kouwenhoven, B. de Jong, P.A. Sillevis Smitt, M.J. van den
Bent, and J.M. Kros; Neuro-oncology, Erasmus University Medical
Center, Rotterdam, The Netherlands
Gliomas are known for their morphologic heterogeneity. Generally, gli-
omas are typed according to areas with classic histological parts, but within
many tumors areas with less typical histology exist. The classic histology of
oligodendrogliomas is the honeycomb architecture with chicken wire-like
vasculature, the tumor cells having a perinuclear shrinkage artifact. How-
ever, tumor parts showing astrocytic differentiation, mixed histology, pre-
dominant gemistocytic parts, pseudoependymal or neurocytic differentia-
tion, rhabdoid changes, or mucoid degeneration are variably encountered in
biopsy material as well. In approximately 60% of oligodendroglial tumors
losses of 1p and 19q are found. This genotype has been linked with a favor-
able response to both radio- and chemotherapy, and patients show better
survival as compared to those suffering from oligodendroglial tumors with-
out these characteristics. It is unclear whether histological heterogeneous
tumors are also genetically heterogeneous, and whether tumor parts other
than those with classic histology will carry the oligodendroglioma-specic
genotype. This information may be important when considering the (high)
probability of sampling error and treatment of these tumors, and glioma
classication in general. For this study a selected group of 14 gliomas,
consisting of 6 anaplastic oligodendrogliomas, 6 mixed anaplastic oligoas-
trocytomas, and 2 glioblastomas, all with partly classical oligodendroglial
and other histology, were used. Within each tumor, up to 8 regions (total
number of regions studied was 60) were characterized morphologically and
genotyped by uorescent in situ hybridization (FISH) for 1p and 19q. Loss
of 1p was found in 8/14 tumors and loss of 19q in 7/12 tumors. Fifty percent
of the losses of 1p were detected in all tumor areas, while the other 50%
were only seen in part of the tumors. In 57% of tumors, loss of 19q was
found in all tumor areas, and in the other 43% only in parts of the tumors.
Loss of 1p was detected in tumor areas other than those with classic oli-
godendroglial histology in 47% of cases, and loss of 19q in 45% of cases.
The results illustrate that, besides phenotypical heterogeneity, there is also
genotypical heterogeneity and that genotyping of gliomas without classic
oligodendroglial histology may result in positive identication of 1p/19q
losses. This may be relevant for small (stereotactic) biopsies. Alternatively,
these ndings may reect limitations of the tests used; therefore, further
research is indicated.
202. A PROSPECTIVE, BLINDED ANALYSIS OF A-PROTEIN
LEVELS IN PEDIATRIC PATIENTS WITH CENTRAL
NERVOUS SYSTEM TUMORS
P. Manley,
1
C. Turner,
1
S. Chi
1
, M. Zimmerman,
1
C. Chordas,
1

A. Gordon,
1
A. Thomas,
1
K. Hoffman,
2
and M. Kieran
1
;
1
Pediatric
Oncology, Dana Farber Cancer Institute, Boston, Massachusetts;
2
Cytra
Corporation, Wrentham, Massachusetts; USA
The purpose of this prospective blinded study was to evaluate the sensi-
tivity and specicity of A-PROTEIN levels in pediatric patients with brain
tumors. A-PROTEIN is a 23 kD molecule that is highly conserved among
mammals and non-mammalian species and was initially named for its pre-
sumed role in adenosine nucleotide exchange. While the exact function of
this protein remains controversial, it does appear to be involved in cell divi-
sion and signal transduction through both G-protein and Ca
2
binding pro-
tein activity. A-PROTEIN is normally localized to the intracellular plasma
membrane. It is located in both normal and neoplastic tissue. Abnormal
expression of A-PROTEIN has been identified in a number of tumors
including small cell carcinoma of the lung, carcinoma of the breast, colon,
prostate, and cervix. Brain tumors have been reported to express exceed-
ingly high plasma levels of A-PROTEIN and thus may be of signicant
diagnostic and prognostic value. Pediatric neuro-oncology patients were
prospectively identied from the neuro-oncology program at Childrens
Hospital Boston and the Dana Farber Cancer Institute. Patients included
those with newly diagnosed disease pre- and post-surgery, during treat-
ment, and during routine follow-up while maintaining remission (based on
concurrent MRI scans), as well as at the time of recurrence or progression.
A total of 154 A-PROTEIN levels in both CSF and blood from 54 pediatric
patients were evaluated. No correlation of A-PROTEIN level was detected
based on patient age (within the pediatric population) or clinical status.
Forty-nine samples from patients with no evidence of disease based on MRI
scans were evaluated. Of these samples, 46% were negative, while 29%
were positive and 25% were equivocal. For patients with stable disease,
73 samples were obtained, and 51% demonstrated a negative A-PROTEIN
level; 19% of these samples were positive and 30% were equivocal. By con-
trast, 20 samples from patients with progressive disease were evaluated. Of
these, 55% had normal A-PROTEIN levels, 35% were positive, and 10%
were equivocal. Based on these results, the sensitivity of the assay was 39%
and the specicity was 62%. Based on our data, A-PROTEIN levels were
not predicative of disease status in children with brain tumors. Whether
this results from the variability of A-PROTEIN levels during growth and
development needs to be further evaluated.
203. RESPONSE TO RADIOTHERAPY IS NOT ASSOCIATED
WITH LOSS OF HETEROZYGOSITY (LOH) FOR
CHROMOSOMES 1p36 AND 19q13 IN WHO GRADE II
ASTROCYTIC GLIOMAS
A. Fathi,
1
C. Murtin,
3
E. Vassella,
3
M. Arnold,
3
J. Curschmann,
2

M. Reinert,
1
P. Siegenthaler,
1
J. Weis,
3
A. Kappeler,
3
and L. Mariani
1
;
1
Universittsklinik fr Neurochirurgie and
2
Universittsklinik fr
Radioonkologie, Inselspital, Bern, Switzerland;
3
Institut fr Pathologie,
Universitt Bern, Switzerland;
4
Universittsklinik der RWTH, Institut
fr Neuropathologie, Aachen, Germany
Loss of heterozygosity (LOH) of 1p36 and 19q13 is probably both prog-
nostic for survival and a predictive marker of chemosensitivity in patients
with WHO grade II gliomas. A high proportion of patients with WHO
grade II oligoastrocytomas (OA) and astrocytomas (A) respond to radio-
therapy. We investigated whether or not the presence of these deletions
might be related to response to radiotherapy in those tumors. From our
database of patients with gliomas WHO grade II (OA, A) treated between
1991 and 2000, we selected patients who received radiotherapy either at
primary diagnosis or after tumor progression. The tumor volume was mea-
sured and monitored after radiation therapy. Tumor response was assessed
according to standard criteria and graded as complete response (CR), par-
tial response (PR, 50%), minor response (MR, 25%), and progressive
disease (PD, 25%). The presence of LOH for chromosome 1p (4 loci) and
19q (3 loci) was assessed on archival, parafn-embedded tumor specimens
by using a PCR-technique. Thirty-eight patients received radiotherapy.
There were 31 A and 7 OA. The tumor volume was available for 27 patients
(71%), 12 having received radiotherapy at primary diagnosis and 15 at
tumor progression. There was no CR. Five patients had a PR (18.5%), 8
patients MR (29.6%), 12 patients a SD (44.4%), and 2 had PD. The LOH
status could be assessed in 17 and 16 patients for 1p and 19q, respectively.
Of 12 patients with intact chromosome 1p36, 6 had either PR or MR, and 6
had either SD or PD. Approximately 50% of patients with astrocytic WHO
grade II tumors show objective tumor response after radiotherapy. Tumor
response is not associated with LOH for 1p36 and 19q13.
204. PROGNOSTIC VALUE OF 1p/19q REARRANGEMENT IN
OLIGODENDROGLIOMAS: RETROSPECTIVE STUDY WITH
86 PATIENTS
Y. Guegan,
1
N. Auger,
2
S. Saikali,
3
A. Hamlat,
1
B. Laviolle,
4
and
M. Le Calve
2
;
1
Clinique Neurochirurgicale,
2
Laboratoire de
Cytogntique,
3
Laboratoire dAnatomopathologie, and
4
Service de
biostatistiques, CHU Pontchaillou, Rennes, France
Oligodendrogliomas represent up to 25% of diffuse gliomas, the most
frequent primary tumors of the central nervous system. The tumors, which
present a 1p/19q co-deletion, have a better prognosis, with a higher chemo-
radiosensitivity and longer survival. A retrospective study of 1p/19q rear-
rangements in primary oligodendrogliomas treated between 1990 and 2001
at the Rennes Hospital was done. A confrontation between clinical data
and factors inuencing the prognosis was made. The tumors were graded
by two neuropathologists. FISH (uorescent in situ hybridization) was
performed on 5-mm-thick sections from archival formalin-xed parafn-
embedded tumors. The probes used, located in 1p36 and 19q13 regions,
were developed from BAC and were directly labeled by nick translation.
For each probe, hybridization signals were scored from a minimum of 200
nuclei. A univariate and a multivariate statistical analysis was performed.
The prognosis was correlated with the age of the patients at diagnosis (odds
ratio 1.1), with the presence of necrosis (odds ratio 13.7), and with
the chromosomal status (odds ratio 0.026). We found 58% (n 59) of
tumors with a deleted status, 22% (n 19) with a disomic status, and 20%
(n 17) with a polysomic status. Tumors with a deleted status had a better
prognosis and a better response to chemotherapy than the 1p/19q disomic
tumors. A third population of tumors had a polysomic status, which cannot
be detected by LOH search and is hardly identied by CGH (comparative
genomic hybridization). This population of patients had a poor prognosis
whatever the treatment was. This study reveals the coexistence of three
independent prognostic factors: age, necrosis presence, and mainly chro-
mosomal status. Even though the deleted population is known, we reveal
for the rst time a new population, the polysomic status tumors population,
which has a very poor prognosis.
205. IMMUNOHISTOCHEMICAL SCREENING BASED
ON GENE EXPRESSION PROFILES PREDICTIVE OF
MENINGIOMA RECURRENCE
W. Hendricks,
1,2
S. Coons,
3
A. Scheck,
1
K.E. Ramsey,
4
and
M. Preul
2
;
1
Neuro-Oncology Research,
2
Neurosurgery Research, and
3
Neuropathology, Barrow Neurological Institute, Phoenix, Arizona;
4
The NINDS/NIMH Microarray Consortium, Translational Genomics,
Phoenix, Arizona; USA
Current clinicopathological models used in the diagnosis, prognostica-
tion, and treatment of meningiomas do not always reliably predict tumor
behavior and patient survival. A pathological tool that better distinguishes
the more aggressive subset of these tumors will allow a more accurate pre-
diction of their behavior, leading to earlier therapeutic intervention and
improved patient outcome. Differential gene expression in 54 meningio-
mas was analyzed by using Affymetrix U133 Plus 2.0 GeneChip Human
Genome microarrays. Expression proles based on histologic grade, pri-
mary/recurrent identity, invasion, and whether the tumor recurred within
a two-year follow-up period were used to identify genes likely to provide
clinically useful diagnostic/prognostic information. Class prediction and
clustering analyses were then performed to evaluate the ability of these
proles to predict clinical parameters associated with the aggressive phe-
notype. Genes in proles were subsequently ltered based on the availabil-
ity of antibodies to their protein products. The diagnostic and prognostic
value of each antibody is being evaluated in up to 1200 tumors on 20 tissue
microarrays. Neither the proles made according to the lone parameters of
grade, primary/recurrent status, and invasion nor the proles made accord-
ing to a combination of these parameters predicted tumor recurrence better
than WHO grade alone. However, a prole based on recurrence on follow-
up reveals distinct molecular signatures for these tumors. These data show
that clinically aggressive meningiomas (as dened by clinical recurrence
within 2 years) share common molecular features to allow for expression
proling for diagnosis and prognostication. Searches of publicly available
antibody databases based on genes whose expression can predict aggres-
sive behavior reveal candidates for further screening with antibodies on
tissue microarrays. Current candidates include CD40, CD96, and SFRP4.
These ndings are being further correlated with histologic grade, subtype,
invasion, and recurrence. The use of microarray-based expression proling
to identify prognostically signicant genes that can then be analyzed by
immunohistochemical screening, a technique readily available in clinical
laboratories, provides a novel tool to signicantly enhance clinical manage-
ment of meningiomas and improve overall patient outcome.
206. TEMOZOLOMIDE (TMZ) AS INITIAL TREATMENT
FOR PROGRESSIVE LOW- GRADE OLIGODENDROGLIOMAS:
TREATMENT RESULTS AND CORRELATION BETWEEN
GENETIC PROFILE AND MGMT PROTEIN EXPRESSION
N. Levin,
1
I. Lavon,
1
B. Zelikovitsh,
1
D. Fuchs,
1
Y. Fellig,
2
F. Bokstein,
1

and T. Siegal
1
;
1
Leslie and Michael Gafn Center for Neuro-Oncology
and
2
Department of Pathology, Hadassah Hebrew University Hospital,
Jerusalem, Israel
Loss of heterozygosity (LOH) on chromosomes 1p and 19q has been
associated with favorable response to chemotherapy and good prognosis
in oligodendrogliomas. The DNA repair enzyme O
6
-methylguanine DNA
methyltransferase (MGMT) may induce resistance to DNA-alkylating
drugs. Recent studies show that TMZ, an oral alkylating agent, has ef-
cacy in progressive low-grade oligodendrogliomas (LGO). Yet, limited
data is available regarding the association between 1p/19q prole and
MGMT protein expression in these tumors. The objective of this study
was to evaluate the response of progressive LGO to TMZ and to assess the
association between 1p/19q prole and MGMT protein expression. Adult
patients whose MRI ndings and/or clinical deterioration were compatible
with progressive LGO were eligible for the study if they were radiotherapy
nave. TMZ dose was 200 mg/m
2
/d for 5 days repeated every 28

days. Clini-
cal and MRI data served for evaluation of outcome, and Kaplan-Meier
estimates were used to assess median time to tumor progression (TTP) and
progression-free survival (PFS). 1p/19q status was evaluated from paired
tumor-blood DNA samples by using PCR-based microsatellite analysis.
MGMT protein expression was studied in parafn-embedded tumor sec-
tions by immunohistochemistry. Twenty-eight patients (median age, 38;
range, 1777) were enrolled. Median time between tumor diagnosis and
TMZ treatment was 33.5 months (range, 1133). Median number of TMZ
cycles/pt was 12 (range, 224). Marked clinical improvement was recorded
in 15 pts (54%), an objective response in 17(61%) with 7 minor responses,
stable disease in 10 (36%), and progressive disease in one patient. Median
TTP is 31 months with PFS of 70% at 24 months. LOH of 1p and/or 19q
was found in 14/16 tumors of whom, 11 improved on TMZ, and 3 had
stable disease. Of the 2 tumors with no LOH, one had stable disease and
the other progressed. MGMT protein expression was evaluated in 16 LGO,
whose LOH status showed 13 codeletions at 1p/19q and 3 had an intact
1p chromosome. LGO with an intact 1p demonstrated high expression
of MGMT (50% nuclear staining) as opposed to tumors with 1p loss
that exhibited a relatively low MGMT (050% nuclear staining). MGMT
expression was signicantly associated with 1p loss (P 0.0004). TMZ is
active in progressive LGO. 1p/19q deletions are associated with response
to TMZ, and MGMT protein expression signicantly correlates with the
allelic loss on 1p chromosome.
207. HISTOPATHOLOGIC GRADING OF ADULT
MEDULLOBLASTOMAS IS NOT PREDICTIVE OF SURVIVAL
M.T. Giordana,
1
B. Pollo,
2
R. Ferracini,
3
A. Silvani,
2
C. DAlessandro,
1

G. Cavallo,
4
A. Paioli,
4
P. Ghiglione,
1
and A. Chio
1
;
1
Department
of Neuroscience, University of Turin, Torino;
2
Departments of
Neuropathology and Neuro-oncology, C. Besta Institute, Milano;
3
Department of Pathology, University of Bologna, Bologna;
4
Department
of Oncology, Bellaria Hospital, Bologna; Italy
Histopathologic grading of pediatric medulloblastomas based upon
increasing grade of anaplasia predicts clinical behavior. The present study
was aimed to grading medulloblastoma of adult patients (18 years old) by
evaluating 83 samples for pathologic features of malignancy and anapla-
sia. Nodularity, desmoplasia, nuclear size, nuclear pleomorphism, necrosis,
endothelial proliferations, and MIB-1 labeling index (LI) value have been
evaluated by multivariate analysis with age, tumor site, and total survival.
Morphometric analysis of nuclear size was performed by using the Eclipse
Net program. Patients treated with standard postoperative radiotherapy (35
Gy to the craniospinal axis and 50 Gy to the posterior fossa) were consid-
ered for correlation with survival. Pathologic data and total survival were
compared by Kaplan-Meier and log-rank analysis. Nodular/desmoplastic
features were present in 50% of cases; however, none of them qualied
for medulloblastoma with extensive nodularity. Small isomorphic nuclei
characterized 70% of tumors, while others had a moderate nuclear pleo-
morphism without signicantly enlarged nuclei. Isolated large cells with
pleomorphic nuclei were found in two cases only. MIB-1 LIs ranged from
9.6% to 55.5% (median 30.2%) and were not signicantly different in nod-
ular versus classic medulloblastomas. No correlation was found between
total survival duration and the evaluated pathologic features. Assessment of
anaplasia grade based either on coexistence or increasing degree of the ana-
lyzed features did not predict outcome. Severe degrees of anaplasia were not
found. Based on our data, the histologic evaluation of medulloblastomas
from adults with respect to features of anaplasia does not give prognostic
information. For a satisfactory stratication of adult patients with medul-
loblastoma, we have to turn to biological and molecular factors and analyze
their prognostic role.
208. RANDOMIZED CONTROLLED TRIAL OF A PATIENT
SPECIFIC REMINDER OF ROYAL COLLEGE OF PHYSICIANS
GUIDELINES FOR COMMUNICATION IN PATIENTS WITH
MALIGNANT GLIOMA
R. Grant,
1
I. Whittle,
1
A. Gregor,
1
G. Kaar,
2
R. Swingler,
3
and P.
Mathew
3
;
1
Edinburgh Centre for Neuro-Oncology, Western General
Hospital, Edinburgh, Scotland;
2
Neurosurgery, Aberdeen Royal
Inrmary, Aberdeen, Scotland;
3
Neurosciences, Ninewells Hospital,
Dundee; Scotland; UK
Some studies have shown that Patient Specic Reminders (PSR) can
improve the standard communication between treating doctors and patients
or colleagues. We developed PSR cards to remind treating doctors of the
Royal College of Physician guidelines on communication, for example,
breaking the news of diagnosis of brain tumor, diagnosis, and prognosis. We
were interested to see if a PSR attached to the patient notes improved com-
munication. This was a randomized controlled trial involving 163 patients
with intrinsic brain tumors treated in three centers (Edinburgh, Aberdeen,
and Dundee), randomized to PSR card to inform the neurosurgeon about
RCP guidelines about communication with patient and colleagues or no
PSR card (standard care). Quality of communication with (a) patient, (b)
GP, and (c) radiation oncologist was assessed after discharge by conden-
tial questionnaires. Completed questionnaires returned by patients were
scored by 3 blinded independent assessors of a brain tumor charity. The GP
questionnaires returned by GPs were sent to 3 independent GPs for scoring;
the Radiation Oncology questionnaires were sent to 3 independent hospital
doctors for scoring. The assessors rated the individual questionnaires for
overall communication as very good, good, poor, very poor. Assessors only
knew the age and the presumed diagnosis. The median score of the 3 asses-
sors was taken as the overall grade for each questionnaire. Statistical analy-
sis was performed by using the chi squared test. Quality of communication
in patient questionnaires was considered good or very good in 55% to 74%
and very poor in 9%. There was no statistical difference between those
allocated a PSR and those with standard care (DF 6, P 0.365). GPs
considered communication good or very good in 36% to 48% of cases and
very poor in 13% to 24% (PSR 13%: standard care 24%), no statistical dif-
ference (DF 6, P 0.53). Hospital doctors considered rated communica-
tion with radiation oncologists as good or very good in 55% to 62% of cases
and very poor in 2.5% to 5%, no statistical difference (DF 6, P 0.965).
This is the only RCT of the effect of a patient-specic reminder regarding
guidelines on communication for neurosurgeons dealing with brain tumor
patients. The PSR alone did not dramatically or statistically signicantly
improve the quality of information given to patients, GPs, or radiotherapists
by the neurosurgical teams in 3 centers in this study.
209. THE COLLECTIVE CARE PATHWAY OF THE NEURO-
ONCOLOGY NURSE PRACTITIONER AND THE ADVANCED
PRACTITIONER IN RADIOTHERAPY
L. Muffett and K. Burton; Oncology, Addenbrookes Hospital,
Cambridge, UK
Primary brain tumors (gliomas) represent a small proportion of all can-
cers, yet the impact of the disease is catastrophic, as the illness causes both
physical and cognitive disabilities. For the client and family, this presents
many challenges, as the journey is unpredictable. It is this uniqueness that
requires specialist input, ranging from advanced technical treatments to
palliative care. The concept of the above roles evolved from The Calman-
Hine Report (DOH 1995) and The NHS Plan (DOH 2000 A), which sub-
sequently initiated The NHS Cancer Plan (2000 B). It was this report that
explicitly identied recommendations for staff investment and patient care
and acknowledged the need for site-specic multidisciplinary teams. It pro-
posed that core members of the team should include a specialist nurse and a
therapy radiographer. Since their implementation, both roles have evolved,
and the current designations replace the previous titles of Specialist Radiog-
rapher and named nurse. Both roles are diverse. The Advanced Practitioner
role focuses on providing expert knowledge in the planning and delivery
of radiotherapy, coordinating the pathway of radiotherapy, and providing
support for the patient and family. Whereas the Nurse Practitioner role is
very subjective, it focuses on providing support to both clients and fam-
ily members at all stages of the journey. The role encompasses a holistic
approach, centering on providing palliative care. To work together autono-
mously, without overlapping, has required setting foundations. Dening
roles, setting boundaries, undertaking clinical supervision, and reecting
on the illness trajectories have assisted us in planning the care pathways. All
patients are discussed within the teams multidisciplinary meeting prior to
their rst consultation. It is at this assessment that planning of the patient
journey begins. Depending on treatment intervention, a determination is
made of which professional takes the lead. Fortunately, due to our profes-
sional exible working relationship, when situations change or remits of
roles are stretched, we are able to adapt accordingly to focus on service
delivery rather than egos! We believe that this unique combined approach
enhances patient care. Both roles provide expert knowledge, which is piv-
otal to the patient journey, the team, and each other. It is this awareness of
roles, competencies, boundaries, and patient preference that allows us to
identify phases of the patient journey which require greater input. In order
to evaluate the impact of our roles on the service, we aim to audit the patient
experience in the very near future.
210. IMPACT OF A NEUROONCOLOGIST- COORDINATED
BRAIN TUMOR MANAGEMENT TEAM ON SURVIVAL
OF PATIENTS WITH GLIOBLASTOMA MULTIFORME:
THE HARTFORD HOSPITAL EXPERIENCE
A. Flowers; Neurology, Hartford Hospital, Hartford, Connecticut, USA
In the United States it is estimated that 18,400 patients are diagnosed
per year with primary malignant brain tumors (BT) (ACS, 2004). Malignant
gliomas account for 77% of malignant primary BT in adults. Over 50%
of patients with malignant glioma have glioblastoma multiforme (GBM).
Most of these patients are cared for in a community setting on a one way
street pathway, from the diagnosing physician to a neurosurgeon, then a
radiation oncologist, then a medical oncologist. In centers where the care of
BT patients is provided by a multidisciplinary team, with either a neuroon-
cologist or a medical oncologist assuming a coordinator role, patients are
more likely to become active participants in their care and to enroll in clini-
cal studies, and the overall patient satisfaction is higher. The present study
evaluates outcomes of patients with GBM cared for at Hartford Hospital,
in a multidisciplinary setting. From 1996 to 2003, 100 patients with GBM
were evaluated at Hartford Hospital by a multidisciplinary team coordi-
nated by a neuroooncologist. Treatment and survival data are available for
76 patients; 24 patients were lost for follow-up. Median age was 59 years
(2487 years). There were 43 males and 33 females. Seventy-six patients
had surgery, 10 had second and 3 had third resection, 76 patients had exter-
nal beam RT, 8 had radiosurgery, and 54 had chemotherapy (11 in clinical
studies). Survival data for these patients was analyzed and compared with
survival of a group of 35 GBM patients (median age 71; 3788) treated at
Hartford Hospital with RT alone or palliative care, either due to patients
age, or poor prognostic factors. Survival was also compared with national
SEER reported data. Survival for patients treated by the multidisciplinary
team (MDT) was signicantly longer than for patients treated conserva-
tively. Median survival was 1 year versus 5.5 months. Comparative 1-, 2-,
5-, and 10-year survival rates are for HH MDT 52.6%, 19.7%, 7.89%, and
1.3%, respectively, versus SEER 29.1%, 8.8%, 3.4%, and 2.4%, respec-
tively. Only 6% of patients treated conservatively were alive at 1 year,
none at 2 years. Functional outcome was also analyzed for MDT-managed
patients. Fifty percent of patients with GBM were still working at 1 year.
Patients who survived beyond 2 years were able to maintain a good level of
functioning up to 4 years. The main predictor for survival was patients age,
with longest survival for patients under 55 years of age. Multidisciplinary
management of patients with GBM, coordinated by a neurooncologist, has
a positive impact on survival by providing comprehensive care; close follow-
up, early intervention at recurrence, more treatment options, and a strong
support system. The conventional pathway is appropriate for patients with
poor performance status, those with rapidly growing tumors, or those who
do not wish to be treated aggressively if at all.
211. THE PRESENTATION OF CHILDHOOD BRAIN
TUMORS: A META-ANALYSIS
S. Wilne,
1
J. Collier,
2
D. Walker,
1
R. Grundy,
3
C. Kennedy,
4
and J. Punt
1
;
1
Academic Division of Child Health and
2
School of Nursing, University
of Nottingham, Nottingham;
3
Institute of Child Health, University of
Birmingham, Birmingham;
4
Department of Child Health, University of
Southampton, Southampton; UK
Brain tumors can be difcult to diagnose. Many of the initial symptoms
and signs are also seen in other more common and less serious childhood
illnesses. The challenge for health care professionals is to identify children
who may have a CNS tumor from the majority that do not. A meta-analysis
of the presenting signs and symptoms of childhood brain tumors was con-
ducted as the initial stage in a project to devise guidelines for the refer-
ral and imaging of children who may have a CNS tumor. The objective
of this study was to conduct a meta-analysis of the presenting symptoms
and signs of childhood brain tumors. The English-language literature was
searched from 1996 to 2004 by using Medline, Embase, and the Cochrane
library. Case-series and cohort studies detailing the presenting symptoms
and/or signs of children with brain tumors and including at least 10 chil-
dren were included in a meta-analysis of presenting symptoms and signs.
Thirty-two papers describing 2200 patients were identied as satisfying the
inclusion criteria. Sixty-eight symptoms and signs were identied as occur-
ring in children with brain tumors, although not every paper contained
data on every symptom and sign. Meta-analyses of the data revealed that
the most frequent presenting signs and symptoms were headache (43%;
41%45%); nausea and vomiting (41%; 39%43%); abnormalities of gait
and co-ordination (29%; 27%31%); papilloedema (17%; 16%19%);
changes in behavior and school performance (13%; 12%15%); seizures
(13%; 12%15%); signs of raised intracranial pressure (11%; 10%13%);
squint (11%; 10%13%); ataxia (10%; 9%11%); and anorexia or weight
loss (8%; 7%9%). Thirty-one additional symptoms and signs occurred
at lower frequencies. This analysis emphasizes the diverse ways in which
childhood brain tumors present. While the classical symptoms of raised
intracranial pressure, headache, and vomiting are identied as the common-
est presenting symptoms of childhood brain tumors, they occurred in fewer
than 50%. Childhood CNS tumors may present in multiple ways and thus
to many branches of medicine. Brain tumors have a long symptom interval
in comparison to other childhood tumors. Families and patients nd this
distressing, and it may adversely affect outcome. Awareness among health
care professionals that the presentation of brain tumors may mimic that of
more common childhood illnesses may facilitate diagnosis and reduce their
symptom interval.
212. THE IMPACT OF THE NURSE PRACTITIONER ON
LENGTH OF STAY OF NEURO- ONCOLOGY PATIENTS
IN AN ACUTE CARE SETTING IN A LARGE TEACHING
HOSPITAL
G.C. Kolak,
1
J.W. Henson,
1
N.M. Leahy,
1
E. Stephen,
2
and C. Pappas;
1
Massachusetts General Hospital;
2
Center for Neuro-Oncology, Boston,
Massachusetts, USA
The value of the nurse practitioner (NP) as part of a multidisciplinary
team has been well documented in both nursing and medical literature.
The Massachusetts General Hospital (MGH) established a full-time nurse
practitioner position to decrease the length of stay (LOS) of neuro-oncology
patients, with the additional goals of improving patient education and out-
patient transition. The neuro-oncology NP program was initiated in 1999.
A retrospective analysis was performed of the average LOS of 627 patients
admitted to the neuro-oncology service for three periods: 8 months prior
to the addition of the NP (period 1) and the two 8 month periods follow-
ing addition of the NP (periods 2 and 3). We also analyzed the number of
LOS outliers between the rst two periods. Data were obtained from the
hospitals Clinical Performance Management (CPM) database. The neuro-
oncology team consisted of a staff neuro-oncologist, neuro-oncology fel-
low, and NP. The NP assisted in the medical management and discharge
planning of general neuro-oncology patients hospitalized due to complica-
tions of their disease or side effects of treatment. The NP also managed all
elective chemotherapy patients by coordinating pre-admission laboratory
evaluation, writing chemotherapy and admission orders, performing daily
medical care, and planning patient discharges. Average LOS was 10.7 days
in period 1, prior to the addition of the NP. There was a decrease in aver-
age LOS of 3.3 days (from 10.7 to 7.4 days) at the end of period 2, and 4.5
days (from 10.7 to 6.2 days) at the end of period 3 (P 0.05). There were
10 non-chemotherapy patients who had LOS greater than 2 SD of average
LOS. Of the 10 patients, 7 were admitted in period 1, prior to the addition
of the NP, whereas 3 were admitted in period 2. Informal interviews with
staff neuro-oncologists, nursing administration, and staff nurses revealed
an improvement in patient education, smoother transitions to the outpa-
tient setting, and better coordination of patient care in general. This sub-
sequently resulted in an overall improvement in patient satisfaction. Nurse
practitioners are effective in the acute care setting in decreasing LOS of
neuro-oncology patients. The addition of the nurse practitioner improved
the care of neuro-oncology patients both by decreasing average LOS and
increasing patient satisfaction.
213. QUALITY OF LIFE IN BRAIN CANCER PATIENTS AND
COPING STRATEGIES
C. Lucchiari, R. Lovati, A. Silvani, M. Eoli, E. Lamperti, A. Salmaggi,
A. Boiardi, and G. Filippini; Istituto Nazionale Neurologico C. Besta,
Neuro-oncology, Milan, Italy
The aim of our study was to measure the quality of life (QoL) in brain
cancer patients using objective and subjective measures, in order to assess
QoL and problematic areas to briey t patients needs in a hospital setting.
We studied 111 consecutive subjects with high-grade gliomas between 23
and 79 years old (mean age, 48.9) during their treatment at the National
Neurological Istitute C. Besta of Milan (Italy). Each patient completed
the following scales: the Fact-G scale 4.0 (Italian version) and the Fact-Br
brain specic module for the evaluation of QoL in brain cancer patients, the
HAD scale for depression and anxiety, and the half-structured interview
SeiQol-Dw for subjective evaluation of QoL and patients experience. For
each patient we rst measured the functional status by the Karnofsky index
(KPS) and the cognitive status through the Mini Mental State Examination.
The mean ( SD) score of the Fact-G was 73.4 13.7 and the Fact-Br was
123.1 23.7. The HAD depression sub-scale showed that 19% of patients
had a light to moderate depression and 3% a severe depression state. Con-
cerning the SeiQol-Dw interview, we performed a content analysis of the
SeiQol descriptions, and we categorized answers in eight main classes of
coping behavior. Most patients found in familial or social support and in
positive actions and thoughts good strategies to cope with the illness. Fur-
ther, women were observed to use spiritual and positive thoughts more
frequently. At the opposite extreme, men preferred more concrete strategies
drawn to improve their functional role in family and society. Males also
reported negative thoughts more frequently than women. The psychological
adjustment to the illness by males seemed to be more difcult. Interaction
between measures showed that global Fact-Br score was better predicted by
Kps and depression, while the SeiQol general score was mainly predicted by
depression scores. Depression did not appear to change in correlation with
age, sex, and time from rst diagnosis. Our data conrm that patients with
high-grade gliomas may cope with the illness without developing severe
depression and anxiety. Though QoL is strongly affected by physical symp-
toms, patients are able to use different strategies to t the situation, during
aggressive therapy, radiotherapy, and chemotherapy. We think that the cop-
ing strategies used by patients help them to adjust to the illness and con-
tinue their treatment in a hopeful and protective context. This indicates the
necessity to sustain patients positive context with an adequate assessment
and comprehension of the potential coping strategy. This could be done by
adopting specic instruments that help to develop an understanding of the
patients subjective experience.
214. PRE-, POST- OPERATIVE AND FOLLOW-UP FMRI
EVALUATION OF CONDITIONAL MOTOR ASSOCIATIVE
FUNCTION IN PATIENTS WITH LOW- GRADE GLIOMAS
C. Amiez,
1
P. Kostopoulos,
1
A. Champod,
1
M. Petrides,
1
R. Del
Maestro,
2
J. Doyon,
3
and L. Collins
4
;
1
Neuropsychology Unit, Montreal
Neurological Institute
2
Brain Tumour Research Centre, Montreal
Neurological Institute and Hospital;
3
Centre de Recherche de lInstitut
Universitaire de Griatrie de Montral;
4
McConnell Brain Imaging
Centre, Montreal Neurological Institute, Montreal; Canada
The goal of this study is to develop a functional Magnetic Resonance
Imaging (fMRI) protocol that assesses higher cognitive functions, other
than language and somatosensory functions, in patients with low-grade
gliomas. These patients have a life expectancy that ranges between 2 and
10 years, and therefore it is crucial that great care be taken to preserve
higher cognitive functions in order to preserve their autonomy and quality
of life post-operatively. In this study, we developed an fMRI protocol for
the pre-operative, post-operative (2 months after the surgery), and follow-
up (8 months after the surgery) evaluation of higher cognitive functions in
patients exhibiting gliomas near the premotor region. In order to assess the
function of the premotor region in these patients, we developed a condi-
tional visuo-motor associative protocol that was tested in 8 healthy control
subjects. In this protocol, the subjects had to associate each one of four
different colors with a specic key press on a mouse with four buttons.
The fMRI results revealed that the premotor region and the supplementary
motor area are specically involved in conditional visuo-motor associative
function. These data demonstrate that the protocol we developed provides
robust and reliable measures of conditional visuo-motor associative func-
tion within the premotor region. We then conducted a pre-operative fMRI
study using this protocol in three patients with tumors near the premotor
region. As in healthy control subjects, pre-operative fMRI data obtained in
these patients showed activity increases within the premotor region. Cou-
pled with the structural MRI, the clinical pre-operative fMRI data were
transferred to an integrated image guided system in the operating room.
The neurosurgeon was able to use the data in the planning of the surgery
in order to spare functional tissue in the premotor region. As a result, the
neurosurgeon was able to optimize the extension of the tumor resection
in the three patients without any decits in the conditional visuo-motor
associative function. In order to study the long-term outcome in terms of
higher cognitive function and the nature of any functional reorganization
that might occur after brain tumor removals, we conducted a post-operative
and a follow-up fMRI scan in these patients. In contrast to the pre-opera-
tive fMRI results, post-operative results showed decreased activity in the
precentral gyrus. The follow-up fMRI revealed activity differences sim-
ilar to the ones observed in the pre-operative fMRI scan. These results
suggested that functional reorganizations occurred following the tumor
resection. The results provided by such preoperative, postoperative and
follow-up fMRI studies are a useful clinical tool for the patients long-term
prognosis.
215. QUALITY OF LIFE IN PEDIATRIC BRAIN TUMOR
SURVIVORS
M.J. Bonner, K.K. Hardy, A.B. Guill, V.W. Willard, K.E. Carter, and
S. Gururangan; Brain Tumor Center, Duke University Medical Center,
Durham, North Carolina, USA
Studies suggest that survivors of pediatric brain tumors may be at risk
for developing symptoms consistent with a nonverbal learning disability
(NLD) as a consequence of their disease and its treatment. Rourke (1988)
described NLD as affecting childrens functioning across multiple domains
including cognitive, academic, and social areas. As a result, individuals with
NLD may experience learning difculties in reading and math comprehen-
sion, math calculation, and written language. In addition, those with NLD
may exhibit decits in social perception, judgment, and interaction skills
that put them at higher risk for social withdrawal or isolation. In contrast
to these weaknesses, individuals with NLD often manifest relative strengths
in rote skills and memorization, verbal processing, reading decoding and
dictation skills. Preliminary data are now available that provide evidence
of the cognitive and academic decits associated with NLD; however, the
social functioning of survivors is not well investigated. The primary objec-
tives of the current study are to demonstrate that a sample of survivors of
pediatric brain tumors have greater impairments in social functioning than
do healthy controls, using both questionnaire and skill assessment data,
and to determine what risk factors are associated with social impairment.
To date, 24 patients (14 females and 10 males) ranging in age from 7 to 16
(M 13.1, SD 2.77) have been enrolled in the study. Diagnoses were
varied, with 8 patients (33.3%) with medulloblastoma, 5 (20.8%) with
ependymoma, 5 (20.8%) with pilocytic astrocytoma, and 6 (25%) with
other tumor types. The majority of patients had been treated with surgery
(n 20, 83.3%), 17 (70.9%) had received hemotherapy, and 14 (58.3%)
had been treated with radiation. Age at diagnosis ranged from 2 to14
(M 6.58, SD 3.20). Participants had been off treatment an average of
5.25 years (SD 2.56; range 111 years) at the time of study. Cognitive
functioning of the sample was variable, with estimated IQs averaging 87.61
(SD 16.18). Broadly speaking, parents rated their childrens social skills
as somewhat lower than would be expected by age-based norms. Subjects
were also rated as having somewhat more social problems, including dif-
culties with nonverbal communication, than would be expected. Contrary
to expectations, parent-reported social problems were not signicantly
associated with age at diagnosis, treatment type, functional or cosmetic
impairment, or estimated IQ scores. However, social problems correlated
signicantly and negatively with scores of performance on a measure of
ability to decode facial emotions. Findings from this study contribute to a
more complete phenotype of the neuropsychological late effects experienced
by survivors and to the development of interventions aimed at ameliorating
disease and treatment-related decits in the growing population of brain
tumor survivors.
216. COGNITIVE FUNCTIONS AND APOE GENOTYPE IN
LOW- GRADE GLIOMA PATIENTS
D. Correa,
1
L.M. DeAngelis,
1
W. Shi,
2
H.T. Thaler,
2
and L.E. Abrey
1
;
1
Neurology;
2
Epidemiology and Biostatistics, Memorial Sloan Kettering
Cancer Center, New York, New York, USA
The purpose of this study was to assess cognitive functions in patients
with low-grade gliomas (LGG) who received radiation, chemotherapy, or
no treatment. We also compared the cognitive performance of patients
who carried the Epsilon-4 allele of the Apolipoprotein E (APOE) gene to
those who carried other APOE alleles. Forty adult patients with LGG and
no evidence of disease progression participated in the study; 16 patients
had received conformal radiotherapy chemotherapy, and 24 patients
had no adjuvant treatment. All patients underwent a neuropsychological
evaluation, and test scores were compared to normative reference groups;
7 composite cognitive domain scores were calculated. APOE genotype was
obtained in 33 patients who were classied in two groups based on the
presence or absence of at least one APOE Epsilon-4 allele. Patients who
received radiation chemotherapy had signicantly lower scores than
untreated patients on the Psychomotor (P 0.03) domain. Analysis of
covariance, adjusting for anticonvulsant regimen (i.e., monotherapy vs.
polytherapy), suggested no signicant adjuvant treatment effect on Psy-
chomotor performance; the results showed that patients on anticonvulsant
polytherapy had lower scores on the Psychomotor domain, regardless of
tumor treatment status. Treated patients obtained signicantly lower scores
than untreated patients on the Non-Verbal Memory (P 0.02) domain.
Analysis of covariance, adjusting for age, showed that patients who com-
pleted treatment at intervals longer than 36 months had signicantly lower
scores on the Non-Verbal Memory domain than untreated patients. Over
60% of treated patients showed mild to moderate white matter conu-
ence on MRI, whereas 90% of the untreated patients had either no or only
minimal white matter changes (P 0.002). Comparisons between APOE
Epsilon-4 carriers (n 8) and noncarriers (n 25) on cognitive domain
scores revealed no signicant differences, but Epsilon-4 carriers had lower
scores on the Verbal Memory domain than did noncarriers. The ndings
suggest that LGG patients treated with radiation chemotherapy had more
difculties in nonverbal memory and were more likely to show white matter
abnormalities on MRI than untreated patients. Psychomotor slowing was
most prominent among patients who were on anticonvulsant polytherapy,
regardless of adjuvant treatment status.
217. THE COURSE OF NEUROCOGNITIVE STATUS IN
HIGH- GRADE GLIOMA PATIENTS
I. Bosma,
1
M. Vos,
1
J. Heimans,
1
M. Taphoorn,
5
N. Aaronson,
6

T. Postma,
1
H. van der Ploeg,
2
W. Vandertop,
3
B. Slotman,
4
and
M. Klein
2
;
1
Neurology,
2
Medical Psychology,
3
Neurosurgery, and
4
Radiation Oncology, Academic Hospital Vrije Universiteit, Amsterdam;
5
Neurology, Medical Center Haaglanden, The Hague;
6
Division of
Psychosocial Research and Epidemiology, The Netherlands Cancer
Institute, Amsterdam; The Netherlands
This work was conducted to study the course of neurocognitive func-
tioning in newly diagnosed high-grade glioma patients and to determine
the tumor, treatment, and patient-related factors affecting neurocognitive
functioning in the course of the disease. Following baseline assessment (i.e.,
after surgery, prior to the start of radiotherapy), follow-up evaluations on
neurocognitive functioning were performed at 8 and 16 months in newly
diagnosed high-grade glioma (HGG) patients and in patients with non-
small-cell lung cancer (NSCLC). HGG patients level of functioning was
compared to the level of functioning in NSCLC patients and to that of age-
and sex-matched healthy controls. A battery of standardized tests was used
to assess neurocognitive functioning. In order to accomplish data reduction,
summary measures were calculated to detect possible decits in the neuro-
cognitive domains of (1) information processing speed, (2) psychomotor
function, (3) attentional functioning, (4) verbal memory, (5) working mem-
ory, and (6) executive functioning. Follow-up data could be obtained in 35
of the 68 HGG patients initially included in the study. Of these, 20 patients
had only one follow-up at 8 months, whereas 15 patients also had a 16-
month follow-up. The patients who also had a follow-up at 16 months had a
better neurocognitive status, were younger, had a signicantly lower tumor
grade, and received lower fraction doses than patients with only an 8-month
follow-up. HGG patients with a shorter follow-up performed worse when
compared to NSCLC patients with the same follow-up. Compared to the
performance of NSCLC patients during the follow-up, no statistically sig-
nicant deterioration was found in neurocognitive function in the course of
the disease in the HGG patients. However, evaluation of HGG patients with
versus HGG patients without tumor recurrence indicated that neurocogni-
tive decline in these patients often precedes clinical signs of tumor progres-
sion. Neurocognitive functioning in HGG patients is mostly affected by
tumor effects and not unequivocally by treatment effects. In the course of
their disease and in the absence of tumor progression, no clear trend toward
further worsening in neurocognitive functioning is to be expected in these
patients. Additionally, neurocognitive function appears to be a prognostic
factor in the course of the disease in these patients.
218. THE END OF LIFE HOSPITAL SETTING FOR
PATIENTS WITH MALIGNANT GLIOMAS
W. Walter, S. Oberndorfer, E. Lindeck-Pozza, H. Lahrmann, W. Struhal,
P. Hitzenberger, and W. Grisold; LBI for Neurooncology, Department for
Neurology, KFJ-Hospital, Vienna, Austria
Despite aggressive treatment, outcome of patients with malignant
gliomas is poor. In the terminal course of the disease, due to social, eco-
nomical, individual, and cultural reasons, some patients are admitted to
hospital care. For the terminal phase, there is a lack of investigations in
the neurological literature. The purpose of this study was to evaluate the
end of life phase in a hospital setting for patients with malignant glioma.
Twenty consecutive patients with malignant gliomas were included in this
analysis regarding symptoms, medication, diagnostic, and interventional
procedures. The last ten weeks before death were divided into three periods:
Period I; from ten weeks to six weeks before death, Period II; six weeks
to two weeks before death; and period III; last two weeks before death.
The patients were comparable regarding age, sex, and overall survival. The
Karnofsky performance scale decreased continuously from period I (aver-
age absolute 70%) to period III (average absolute 10%). Relevant clini-
cal complications, medications, and diagnostics as well as interventional
procedures increased from period I to period III. For the period I symp-
toms of headache and seizures, treatment with antiepileptic drugs as well
as steroids and analgesics was prescribed. Diagnostic procedures such as
MRI, blood tests, or X ray of the chest, as well as interventional procedures
(e.g., urinary catheter, intravenous drug administration), were less frequent
compared to period II or III. In the last period, brain edema, fever, somno-
lence, and pneumonia were the most prominent clinical features. Almost all
patients in period III received transdermal or subcutaneous opioids, uid
replacement, anticoagulation, intermittent oxygen, anticonvulsants, gastric
protection, and urinary catheters, as well as a pressure relief mattress. The
majority of patients died due to infection or respiratory distress. Provid-
ing adequate palliative care to dying patients with malignant gliomas is an
important aspect of treatment. Our study demonstrates that the end-of-life
phase of brain tumor patients has several periods. Contrary to the limited
therapeutic implications, the number of therapeutic and diagnostic inter-
ventions increases toward the terminal phase. Future patient management
needs to be more symptom-oriented and palliative.
219. PATIENT-HELD RECORDS: A PROSPECTIVE STUDY
TO EVALUATE THE USE OF A STEROID BOOKLET BY
PATIENTS WITH MALIGNANT INTRACRANIAL GLIOMA
(MIG)
J. Bentley,
1
G. Hendry,
2
and P. Kane
2
;
1
Palliative Medicine;
2
Neurosurgery, James Cook University Hospital, Middlesbrough, UK
High-dose steroids are commonly used in the management of MIG and
can be associated with signicant side effects. This study investigated the
use of a patient-held record as a possible approach to improving patient
understanding of their medication and monitoring usage. The booklet was
developed by consulting best current evidence, relevant health profession-
als, patients, and their carers. It provided information on steroids, side
effects, and a chart to monitor treatment. Patients were recruited within
one week of the diagnosis of MIG. All had been given verbal information
about steroids by their clinician. Together with their main carer they com-
pleted a questionnaire to determine steroid knowledge. Patients were then
randomized to receive the steroid booklet or not. Four months later they
completed the same questionnaire again and also another to determine
patient/carer satisfaction with steroid use. Twenty-one patients have been
recruited to the study (M 14, F 7, age range, 3875 yrs), and 13 have
completed. Four patients did not complete (2 deaths, 2 disease progression).
Fifty-four percent of the patients managed their own medication, and in the
remainder it was handled by the carer. The mean steroid knowledge score
at recruitment was 50%, and at follow up the mean score was 64%. There
was no signicant difference in the score between the groups. Improved
score was due mainly to increased knowledge of the common side effects
(somatic changes, sleep disturbance). Twenty-three percent of the patients
were dissatised with the amount of written information given about ste-
roids. None of these patients received the steroid booklet. Forty-three per-
cent of the patients with the steroid booklet did not take it to consultations.
All patients with the booklet reported that they had read it and found it
useful. The study points to the important role of carers in patients with
MIG. Preliminary results suggest that a steroid booklet does not improve
patient/carer knowledge about steroids. However, the steroid booklet was
welcomed by patients and their carers as a source of written information.
220. NEUROCOGNITIVE IMPROVEMENT FOLLOWING
SURGICAL RESECTION IN ADULT PATIENTS WITH
PRIMARY BRAIN TUMORS
E. Farace, M. Mut, and M.E. Shaffrey; Neurosurgery, University of
Virginia, Charlottesville, Virginia, USA
The potential for surgical resection to increase or induce neuropsycho-
logical impairment in patients with primary brain tumors is clear. Analyses
of the neurocognitive effects of treatment (radiation or chemotherapy) are
complicated by the unknown neurocognitive effects of surgery. However,
surgery may be the rst and best treatment option, particularly in patients
with meningiomas and other accessible tumors. Therefore, this pilot analy-
sis of patients who underwent neuropsychological evaluation pre-surgery,
and 6 weeks post-surgery, was undertaken to examine the effects of surgery
in this population. Twenty-ve patients with primary brain tumors who
were clinically referred for baseline and post-operative neuropsychological
evaluation were given a standard neuropsychological assessment battery
measuring a broad range of domains (attention, memory, executive func-
tion, visual and verbal memory, psychomotor speed, depression). Scores
were adjusted for patient age and education as norms were available. Scores
were transformed into Z-scores to allow for ease of comparison across tests.
Fifteen patients had a meningioma, and 10 patients had some form of glioma
(grades 1IV). Eighty-four percent of the patients showed improvement of
at least one standard deviation, in at least one neurocognitive domain, at
6 weeks post-surgery. Thirty-six percent worsened by at least one stan-
dard deviation in at least one neurocognitive domain (percentages overlap
because of some patients who improved in one domain and worsened in
another). Language ability, when impaired at baseline, was most likely to
improve post-surgery. Memory was the least likely neurocognitive domain
to improve. Surgery was found to be, in certain conditions, benecial for
patients neurocognitive function. Although surgery has the potential to
induce decits through focal damage to surrounding tissue, increased risk
of hemorrhage, etc., it may also improve function in some patients, perhaps
through resolution of mass effect, relief of intracranial pressure, or even a
benet of debulking. A better understanding of the neurocognitive effects
of surgical resection is important as patients face decisions regarding their
resection, and to aid researchers in delineating neurocognitive effects of
treatments.
221. INTRACRANIAL MENINGIOMAS IN THE 8TH AND
9TH DECADES OF LIFE: RETROSPECTIVE ANALYSIS OF
SURGICAL OUTCOME IN A CONSECUTIVE SERIES OF 70
PATIENTS
G. Zona, D. Criminelli Rossi, G. Spena, and R. Spaziante; San Martino
Hospital, Neurosurgery, Genova, Italy
Long-lasting life expectancy, improved quality of life, and extensive
availability of neuroradiological facilities (mainly CT scan and MRI) have
dramatically increased the diagnosis of intracranial meningiomas in elderly
population in the last years. As a consequence, more and more neurosur-
geons have to face up to these slow-growing benign lesions whose surgi-
cal management in elderly patients remains quite controversial as the risk/
benet ratio is not always evident. In a consecutive series of 257 intracra-
nial meningiomas operated on at the Department of Neurosurgery of the
University of Genoa (Italy) between December 1995 and January 2004, we
identied a subgroup of seventy patients older than 70 years. Age ranged
from 70 to 86 years (mean, 75.6 4.2), with an evident female prepon-
derance (51 cases). Preoperative clinical status, expressed respectively as
Karnofsky performing scale (KPS) and ASA risk, tumor size and location,
extent of removal, age, and pathology were matched with the early outcome
and, by means of Kaplan-Meier curves, with disease-free survival (DFS) at
long-term follow-up. Mortality was 5.7% (4 cases). Neurological status
improved in 25 patients and remained unchanged in 33 patients (35.7%
and 47.1%, respectively). Eight patients worsened (11.4%); among them,
three patients completely recovered after two weeks and two patients at
long-term follow-up. Eleven patients presented medical complications, and
four patients need re-operation: All of them completely recovered. Long-
term follow-up ranging from 6 to 86 months was obtained in 53 patients
(80.3%) out to 66 surviving patients. Four more patients died, raising the
overall mortality to 11.4%. Neurological outcome, compared with the early
postoperative period, improved in 9 patients (16.7%), remained stable in
38 patients (71.7%), and worsened in two cases (3.7%). None of the prog-
nostic factors considered reached statistical signicance. Nevertheless, sub-
total resection showed to be slightly better than gross total removal in the
Kaplan-Meier survival function. In conclusion, attentive selection criteria
make surgery feasible with acceptable risks and good overall outcome in
elderly patients. In our opinion, for dealing with slow-growing lesions, a
mandatory aggressive attitude is not always justied, since subtotal resec-
tion, especially for a difcult location, to relieve mass effect can produce
better results.
222. DETECTION OF SPECIFIC EXECUTIVE DEFICITS
USING AN ECOLOGICAL BATTERY IN FRONTAL LOW-
GRADE GLIOMAS
M. Roca,
1
T. Torralva,
1
L. Vita,
1
A. Lacroze,
1
M. Calcagno,
1
F. Manes,
1

and B. Diez
2
;
1
Cognitive and Behavioural Neurology Section and
2
Neuro
Oncology Section, Raul Carrea Institute for Neurological Research
(FLENI), Buenos Aires, Argentina
The most common cognitive symptoms of frontal lesions are impaired
executive functions, social interaction, and personal regulation. However,
this form of mental impairment is sometimes so subtle in daily activities
and unsusceptible to testing that these patients seem intellectually intact.
Decits are not found in standard tests because they do not address the
component of real life decision-making and social cognition. Therefore,
the objective of this investigation was to detect specic executive decits in
patients with frontal low-grade gliomas (FG), using an ecological execu-
tive battery, that consists in real-life tests shown to be sensitive to damage
to the prefrontal cortex (PFC). Patients with FG (n 5) were compared
with a group of normal controls (n 5). All patients underwent a standard
neuropsychological examination and the ecological executive battery.
The ecological executive battery used in this study consists of ve tests.
(1) Frontal Assessment Battery (FAB): This consists in 6 subscales exploring
frontal lobe functions. (2) IOWA Gambling Task (IGT): This test simulates
personal real life decision-making activities. (3) Faux Pas Test: The subject
is shown ten faux pas stories and ten stories without a faux pas; the sub-
ject is asked if something was said inappropriately and why. (4) The Hotel
Task: It comprised six distinct activities that would plausibly need to be
completed in the course of running a hotel. (5) METHv: The purpose of
this test, undertaken in the hospital and its surroundings, is to carry out
12 subtasks in a real life situation. Signicant differences were found
between FG and normal controls in both Trail Making Tests (P 0.14 and
P 0.27), Faux Pas (P 0.007), and total scores of the IGT (P 0.034).
In addition, tendencies were noted in the Met hv total error score, which
were not statistically signicant, probably because of the limited size of the
sample. These patients with low-grade gliomas in the frontal cortex did not
exhibit the most typical disinhibited and socially inappropriate behavior,
usually seen after frontal injury. However, the decits in social interac-
tions and decision making were captured by this new ecological executive
battery. The present study has implications for the clinical assessment of
the real-life problems faced by these patients, both within the family and
whenever they might return to work.
223. ENHANCING COPING THROUGH NEURO- ONCOLOGY
PATIENT EDUCATION MATERIALS
R. Calhoun-Eagan, D. Allen, C. Lentz, S. Jackson, J. Blivin, B. Guill, H.
Quinn, S. Boulton, J. Cahill, and P. Lyons; Brain Tumor Center, Duke
The diagnosis of a high-grade glioma forces the patient and family to
make abrupt treatment decisions based on complex choices. At a time of cri-
sis, the steep learning curve overwhelms many. While learning is enhanced
by easily understandable information, not all patients or caregivers pro-
cess information in the same manner. As more oncology offices prefer
that specialized care be coordinated by neuro-oncology referral centers,
the need for proactive communication and effective teaching strategies has
increased. The Brain Tumor Center at Dukes response to this dilemma
has been the development of a multi-modal approach to patient education.
Designed to maximize comprehension and facilitate coping, it is theorized
that individualized chemotherapy teaching sessions with the patient and
caregivers are reinforced by the provision of a comprehensive patient educa-
tion notebook. Since these sessions occur infrequently due to great travel
distances, the notebook has been designed to orient patients to services and
resources and to promote communications with the tertiary center. This
educational tool has been utilized for the past two years with modications.
It is necessary to evaluate the tool to gain insight into its usefulness. A multi-
disciplinary education committee designed the notebook to address specic
treatment information, clinical trials, early detection and management of
complications, keeping track of symptoms, when to call, self-care, center-
specic resources and national glioma resources, and advance directives. A
survey was designed to measure self-condence in coping with therapy and
overall satisfaction with the notebook. The 5-min survey was given to the
patient/caregiver at the clinic encounter after completion of their rst cycle
of chemotherapy. It is expected that 60 patients will be surveyed through a
6-month interval. To date, survey results (n 15) suggest that patients and
caregivers nd the notebooks contents to be comprehensive, useful, appro-
priately tailored to their needs, and helpful in promoting a sense of control
and condence. Items cited as particularly helpful include fact sheets on
treatment side effects, possible complications and seizure management.
Reinforcing individualized educational sessions with customized informa-
tion in a formulated notebook for ease of information processing promotes
communication, condence, and patient empowerment.
224. NEUROPSYCHOLOGICAL IMPACT OF BONE MARROW
OR HEMATOPOIETIC STEM CELL TRANSPLANTATION:
A PROSPECTIVE STUDY IN PATIENTS WITH
HEMATOLOGICAL MALIGNANCIES
H. Harder, M.J. van den Bent, A.R. Van Gool, J.J. Cornelissen, W.
Eijkenboom, R. Barge, and H.J. Duivenvoorden; Neuro-oncology,
Psychiatry, Hematology, and Radiotherapy, Daniel Den Hoed Cancer
Center, Rotterdam; Hematology, Leiden University Medical Center,
Leiden; Psychology and Psychotherapy, Erasmus University, Rotterdam;
The Netherlands
Bone marrow or hematopoietic stem cell transplantation (SCT) is a cur-
rent cancer treatment for patients with malignant hematological disorders.
Better patient selection and development of reduced-intensity preparative
regimens and new transplant techniques have expanded the use of SCT.
SCT is preceded by the use of high-dose chemotherapy with or without
total body irradiation (TBI) to eradicate the malignant disease and suppress
the immune system to allow engraftment of the donor (or autologous) stem
cells or bone marrow. The complications associated with SCT treatment
are signicant due to severe toxicity associated with myeloablative therapy
(including central nervous system toxicity), the period of profound immu-
nodeciency, and the risk at graft failure or graft-versus-host reaction. The
neurotoxic side-effects on cognitive functioning and the consequences on
patients quality of life are major concerns. Cognitive decits following
SCT have been documented in subgroups of patients. Unfortunately, no
attempt has been made to evaluate a progressive decline or stability in cog-
nitive functioning prospectively. The purpose of this study was to address
the extent of cognitive changes associated with SCT in adult patients with
hematological malignancies. A standardized neuropsychological test-
battery assessing multiple cognitive domains was administered to a longitu-
dinal cohort of 101 SCT patients before undergoing SCT (T1) and at 8 (T2)
and 20 months (T3) after baseline. To control for SCT treatment, a refer-
ence group of 82 hematological patients treated with conventional systemic
chemotherapy and/or involved-eld radiotherapy was included. Effects of
subjective cognitive functioning, quality of life (QOL), fatigue, and psy-
chological functioning were measured with ve self-report questionnaires.
Results were compared to normative data. Analysis employed random
regression modelling (RRM). No between-group differences were found
in cognitive functioning at baseline. Changes over time were observed in
attention (P .01) and psychomotor functions (P .03) with poorer func-
tioning in SCT patients. Performance on verbal memory, visual memory,
and visuospatial functions remained stable at follow-up. Negative effects of
gender and age were found, suggesting poorer performance in, respectively,
females and older patients. Positive effects of education were observed in
all cognitive domains, reecting that patients with higher educational had
better test results. Impaired cognitive functioning in SCT patients was
weakly correlated to mental fatigue, reduced motivation, and anxiety at
follow-up. More cognitive decits were observed in patients treated with
TBI, in patients who received prednisone, and in patients who experienced
long-term infections. Intensive myeloablative cancer therapy had an adverse
impact on cognitive functioning over time, in particular, on psychomotor
functions and attention.
225. LATE EFFECTS IN YOUNG ADULTS SURVIVING A
CHILDHOOD PRIMARY TUMOR OF THE CNS
M.J. Verstegen,
1
R. Bem,
2
S. Leenstra,
1
C. Bos,
2
and D.A. Bosch
1
;
1
Neurosurgery, Academic Medical Centre;
2
Pediatric Oncology, Emma
Childrenhospital, Amsterdam; The Netherlands
Central Nervous System (CNS) tumors account for about 20% of all
childhood tumors. They are second in incidence only to leukemia. As treat-
ment results have improved signicantly, leading to an increased number
of patients surviving over the last decades, attention is drawn toward the
long-term effects and quality of life of these patients. In order to monitor
the late effects of childhood cancer treatment, a long-term follow-up clinic
was founded at the Academic Medical Centre in 1996. All patients had
successfully completed their treatment 5 years before they were transferred
to the long-term follow-up clinic. Participants in this study had to be aged
16 years or older and had to be treated for a primary malignancy of the
CNS at childhood. The study included a questionnaire and interview for
psychosocial and educational functioning, a physical examination, and a
laboratory screening of endocrine-axis. Of 61 patients, the late effects were
divided into 11 different groups: endocrinological, fertility, neurological,
second malignancy, dermatological, psychological, orthopedic, hearing
deciency, urologic, visual deciency and a rest group. The most serious
late effects consisted of endocrine dysfunction comprising solitaire growth
hormone (GH) deciency (n 13 [21%]) or a GH deciency combined
with a primary (n 3 [5%]) or secondary hypothyroidism (n 5 [8%]),
late effects associated with fertility occurring in 15 (24%) patients. In four
patients (15%), a secondary malignancy occurred. In total, 42 patients had
a mild (70%), severe (22%), or total (8%) alopecia, of which 23 patients
received a combination of radiation and chemotherapy. The psychological
problems consisted of a broad variety of social and cognitive disabilities.
Twenty patients reported a learning disability, in fteen cases leading to a
decreased level of education compared with the pre-treatment level. Ten
patients attended a special school for learning disabilities. Nine patients
(15%) were treated by a psychiatrist or psychologist. Four out of seven
patients (57%) who received carboplatine experienced hearing loss. The
total number and severity of the late effects exceeded our expectations, and
they should play a role in the assessment of future treatment strategies.
226. DEVELOPMENT OF NEW FMRI AND INTRA-
OPERATIVE TOOLS FOR NEUROSURGERY GUIDANCE
IN PATIENTS WITH BRAIN TUMORS INVADING THE
PARIETAL LOBES
A.S. Champod,
1
C. Amiez,
1
P. Kostopoulos,
1
R. Del Maestro,
3

M. Petrides,
1
J. Doyon,
4
and D.L. Collins
2
;
1
Neurology and
2
McConnell
Brain Imaging Centre, Montreal Neurological Institute, Montreal;
3
Brain
Tumour Research Centre, Montreal Neurological Institute and Hospital,
Montreal;
4
Centre de Recherche de lInstitut Universitaire de Griatrie de
Montral, Montreal; Canada
The objective of this study is to develop new pre-operative neuroimag-
ing and intra-operative behavioral tools for the assessment of high-order
parietal functions in patients with brain tumors. Pre-operatively, we used
an fMRI (Functional Magnetic Resonance Imaging) protocol to assess
mental rotation function in three patients with brain tumors invading the
parietal lobes. We aimed to localize functional regions surrounding the
tumor that were involved in the task. The pre-operative fMRI showed that
three regions were critically involved in this task, that is, the superior and
inferior parietal lobules, as well as the cortex surrounding the intraparietal
sulcus. These fMRI data were then transferred to a neuronavigation sys-
tem to provide direct intra-operative guidance to the neurosurgeon. Using
surgical navigation, the neurosurgeon precisely mapped out in the oper-
ating room not only the location of the tumor but also the critical areas
involved in the mental rotation task. The neurosurgeon used the functional
data to plan the neurosurgery and to determine the safest surgical route
for the tumor resection of each patient. It was therefore possible, with the
use of this technology, to remove as much of the tumor as possible while
preserving critical areas in each patient. A standardized intra-operative pro-
cedure was also developed. During the operation, the patients were tested
on the mental rotation protocol at regular intervals and at specic stages of
the surgery. The online assessment of performance during surgery allowed
the neurosurgeon to verify whether the cognitive and behavioral processes
for mental rotation were preserved throughout the surgery and to adapt the
surgical approach in order to minimize as much as possible the potential
decits post-operatively. Using this procedure, the neurosurgeon spared
the areas that were critical for mental rotation in each patient. This was
reected in stable performance of the patients on the protocol during sur-
gery. The results suggest that this procedure is of great clinical value. It
provides the neurosurgeon with the possibility to balance the benets of
a complete tumor resection with the possible post-operative functional
decits. Ultimately, it allows an optimal tumor resection with the minimal
post-operative neurological decits. The use of this new procedure could
therefore become crucial for the patients post-operative quality of life and
autonomy.
227. PALLIATIVE CARE IN BRAIN TUMOR PATIENTS:
COMPLICATIONS AND SUPPORTIVE THERAPY IN 215
PATIENTS ASSISTED AT HOME
A. Pace, M. Giovannelli, P. Calabretta, M. Di Lelio, C. Parisi, O. Lembo,
F. Guastella, S. Vita, C. Carapella, and A. Pompili; Regina Elena
National Cancer Institute, Rome, Italy
Although the poor prognosis of malignant brain tumors has not been
substantially modied in the last years by anticancer treatment, palliative
care in neuro-oncology received very little attention. Since October 2000 at
the Regina Elena National Cancer Institute of Rome, we started a palliative
home-care program for patients affected by malignant brain tumor after
hospital discharge, with nancial support of Regional Health System. The
aims of this model of assistance are to meet the patients need of care during
the evolution of the disease, to provide rehabilitation at home, to improve
the patients quality of life with palliative care, and to facilitate death at
home. Neuro-oncologic home staff includes 1 neurologist, 5 nurses, 2
rehabilitation therapists, and 1 psychologist. In the rst three years of our
program 215 patients have been assisted at home and 131 died. The compli-
cations in the last phase of disease were pulmonary infections (10.6%), deep
venous thrombosis (9.7%) with embolic complications in 6 cases, diabetes
due to chronic steroid treatment (8.4%), and psychiatric syndromes (5.7%).
Seventy-nine patients (37%) presented epilepsy despite anticonvulsant treat-
ment; 24% presented adverse effects to medication (chemotherapy, antiepi-
leptic drugs, and steroids). Sixty-nine percent (90/131) of the patients were
able to die at home. Among the 131 patients who died, the most frequent
symptoms in the terminal phase were lethargy (35.5 %), dysphagia (31.8
%), and headache (12.3 %). A cost/utility analysis showed that as a group,
for the patients receiving continuing home care, the hospital readmission
rate and the median time spent in hospital in the last four months of life
are signicantly lower than in a control group (P 0.01). Future clinical
research strategies should include a new model of care for brain tumor
patients, with special attention to palliative home-care models.
228. EXTENDED ABSTRACT: PRIMARY CENTRAL
NERVOUS SYSTEM LYMPHOMA: RECENT ADVANCES IN
DIAGNOSIS AND TREATMENT
N. Arita; Department of Neurosurgery, Hyogo College of Medicine,
Nishinomiya, Japan
Primary central nervous system lymphoma (PCNSL) has been con-
sidered to be a rare tumor, but most literature published since the 1980s
has reported that PCNSL has been increasing in frequency. It now aficts
approximately 300 people in Japan and 1000 in the United States each
year.
The pathogenesis of PCNSL remains obscure. It arises in the central
nervous system where no lymphocytes are present in the normal state.
Most PCNSL is B-cell derived non-Hodgkins lymphoma (NHL). Patients
frequently have multiple lesions in the brain. Compared to noncentral
nervous system (CNS) NHL, PCNSL is refractory to any treatment, and
prognosis is dismal. Primary central nervous system lymphoma often
relapses within the radiated eld in which a dose sufcient to control non-
CNS NHL is given. Primary central nervous system lymphoma arises in
both immunocompetent and immunocompromised populations. These
two groups present different clinical features. In this report, PCNSL in an
immunocompetent population is described.
Primary central nervous system lymphoma affects any age, with a peak
incidence in the fth to seventh decade. Recently, PCNSL arising in the
elderly seems to be increasing. Slight male preponderance has been noted
at all ages. Primary central nervous system lymphoma has no specic diag-
nostic features. It may exhibit various clinical presentations, including
increased intracranial pressure, focal neurological symptoms, and demen-
tia. Uveitis is not rare as an initial symptom. Primary central nervous system
lymphoma arises anywhere in the brain. Most common sites are the peri-
ventricular and cortical regions. The tumor is multifocal in 20% to 30% of
cases. Unlike glioblastoma, which typically shows ringlike enhancement in
CT and MRI, PCNSL exhibits circumscribed homogeneous enhancement.
Peritumoral edema is common. Calcication, cyst formation, hemorrhage,
and necrosis are not accompanied.
Histopathological conrmation is essential to establish diagnosis and
determine treatment strategy. Stereotactic biopsy is currently the diagnostic
procedure of choice. Total resection does not correlate with prolongation
of survival in PCNSL. Therefore, extended resection of the tumor, enhanc-
ing the risk of new neurological decits, is not warranted in most patients.
Steroids should be withheld before biopsy because the tumor may disap-
pear completely. T-cellderived PCNSL has been rarely reported. But, the
vast majority of PCNSLs correspond to B-cell NHL and are categorized
into the subtype of diffuse large cell lymphomas. Immunostaining using
various antibodies is required for the nal diagnosis. Primary central ner-
vous system lymphoma differs from systemic lymphomas in several aspects.
However, until now, no evidence supporting that PCNSL is biologically or
genetically different from systemic lymphomas has been proposed. From
cytological and genetic observations, CNS B-cell-derived diffuse large cell
lymphomas are related to lymphocytes in the germinal center in the dif-
ferential stage.
Primary central nervous system lymphoma had been an incurable dis-
ease. If untreated, median survival time (MST) of patients was reported to
be two to three months. Surgical resection alone did not provide any benet
to patients with MST of one to four months. The object of the surgery is to
conrm histological diagnosis. Forty percent of PCNSL regresses with ste-
roids, but duration of the tumor remission is several weeks. Primary central
nervous system lymphoma is very sensitive to irradiation, and response rate
is 60%. However, PCNSL soon recurs, and radiotherapy increased MST to
only 10 to 18 months. There is no evidence that doses greater than 40 Gy to
the tumor improve survival. The predominant cause of treatment failure is
the recurrence of the tumor within the brain.
Chemotherapy has been applied to PCNSL since more than 20 years
ago. Agents having the most effective antitumor activity against non-CNS
NHL are anthracycline and cyclophosphamide. Combined regimens such
as CHOP that include these two agents have been established as a standard
treatment of choice in non-CNS NHL. These two agents cannot cross the
blood-brain barrier. When patients with PCNSL were treated by CHOP or
CHOD, the MST was 45 weeks to 16 months, not exceeding the result of
radiotherapy alone. Since the early 1990s, preirradiation high-dose metho-
trexate (HD-MTX) was reported to produce a higher response rate as well
as prolonged survival. Subsequently, several clinical trials were published to
suggest the superiority of preirradiation HD-MTX over radiotherapy alone.
High-dose methotrexate followed by radiotherapy brings a response rate of
80% to 90% and MST of 30 to 40 months. Long-term survival or even cure
can be expected in a limited number of patients. Clinical results supported
that HD-MTX followed by irradiation produces better treatment results
than irradiation followed by chemotherapy. Any combined chemotherapy
without HD-MTX did not surpass HD-MTX. Now, systemic HD-MTX
followed by whole cranial irradiation is recommended as a standard treat-
ment. These reports were phase 2 trials, and efcacy of HD-MTX has not
been validated by randomized prospective phase 2 studies. Because of the
small number of patients and large difference of survival between these
two treatments, phase 2 study has not been accepted to perform by most
physicians.
The molecular weight of MTX is 454, and MTX is not lipid soluble.
When given in a low dose, MTX cannot cross the blood-brain barrier. When
a dose greater than 50mg/kg or 1g/m
2
is administered, the cerebrospinal
uid (CSF) level of MTX was demonstrated to elevate over 1 M, which has
an antitumor effect against lymphoma cells. In most clinical trials, high-
dose is dened as greater than 1g/m
2
of intravenous MTX. Optimal dose
and number of cycles of HD-MTX has not been established. We reported a
single institutional trial of HD-MTX followed by whole brain irradiation.
Median survival time and median relapse-free survival were 39.3 and 35.2
months, respectively, for 28 assessable patients. Response rate was 93.8%
in rapid (three-hour) infusion and 58.3% in regular (six-hour) infusion.
Rapid infusion produced a higher level of MTX in the CSF and signicant
tumor volume reduction. Thus, HD-MTX improved the prognosis of the
patients with PCNSL; however, the ve-year survival rate still remains 20%
to 25%. High-dose methotrexate is not effective in all cases. A few negative
trials were also reported. Why some tumors are resistant to MTX has not
been claried. For relapsed or refractory tumors, there is no established
standard treatment.
For further improvement, new trials are being tested. Combined chemo-
therapy trials based on HD-MTX have been reported with MST of up to 60
months. High-dose combined chemotherapy with bone marrow or stem cell
transplantation was also reported. The role of intrathecal chemotherapy
to PCNSL is not clear. New therapeutic modalities have been tested. In
systemic NHL, response rate to the humanized anti-CD20 antibody ritux-
imab is 50%. Most PCNSL expresses the cell surface molecule CD20. If
rituximab(146kDa) is administered intravenously, its CSF levels are very
low. Intrathecal rituximab may be of advantage. Currently, there is limited
evidence for effectiveness of rituximab against PCNSL. Temozolomide, an
alkylating agent recently used to treat gliomas, has a modest but true activ-
ity against PCNSL. These agents have been recently used in relapsed or
refractory tumors after HD-MTX as a salvage therapy.
Delayed neurologic toxicity due to a combination of HD-MTX and
whole brain irradiation has been recognized. As survival is prolonged by
chemotherapy, this complication overshadows quality of life, especially in
the elderly. Patients with this complication present cognitive dysfunction,
dementia, and ataxia. In MRI, diffuse brain atrophy and leukoencepha-
lopathy are observed. Delayed neurotoxicity is more frequent in patients
older than 60 years. To reduce this complication, several trials have been
reported. Radiotherapy is excluded in aged patients or deferred in patients
with complete response with chemotherapy.
In immunocompetent persons, PCNSL will be expected to be more
common in the country where the aged population further increases. Basic
research to understand the pathogenesis of PCNSL is mandatory. Multi-
institutional prospective trials will ensure the establishment of evidence-
based standard treatment strategies.
229. EXTENDED ABSTRACT: NOVEL THERAPIES AGAINST
PRIMARY CNS LYMPHOMAS
A.J.M. Ferreri

and M. Reni; Medical Oncology Unit, Department of
Oncology, San Raffaele H. Scientic Institute, Milan, Italy
Despite recent progress, primary central nervous system lymphomas
(PCNSL) still exhibit one of the worst prognoses among non-Hodgkin
lymphomas (NHL). Chemotherapy followed by radiotherapy is the most
commonly used strategy. The most effective drug is high-dose methotrexate
(HD-MTX) (response rate: 52%100%; two-year overall survival: 58%
72%), while chemotherapy regimens without this drug comprehensively
do not perform any better than radiotherapy alone (Ferreri et al., 2003a).
Several attempts to improve outcome by adding other drugs, empirically
chosen on the bases of extracerebral NHL experience or of the capability to
permeate the blood-brain barrier (BBB), to HD-MTX have been performed.
However, only a few drugs had been previously evaluated as single agents in
phase 1/2 trials, in patients with relapsed or refractory PCNSL. New strate-
gies aimed to intensify chemotherapy as well as to replace consolidation
radiotherapy are now a matter of investigation. Moreover, investigators are
focusing on improving drug bioavailability in different areas of the CNS,
such as eyes and meninges. This paper summarizes new drugs and strategies
against PCNSL and discusses their current role and future developments.
New Drugs: The reduced number of available active drugs limits further
improvements in chemotherapy efcacy, which remains the most pressing
issue in PCNSL. Preliminary results from small phase 2 studies in relapsed
patients are now available with temozolomide, topotecan, and rituximab,
and some retrospective evidence suggests that the addition of high-dose
cytarabine to HD-MTX could be associated with survival improvement
(Ferreri et al., 2002).
Temozolomide is an oral second-generation alkylating agent that spon-
taneously undergoes chemical conversion to MTIC (5[3methyl-1triazeno]
imidazole-4carboxamide), resulting in O-6 methylguanine-DNA methyl-
transferase depletion. This drug has been associated with excellent toler-
ance and a 26% overall response rate, mostly complete remissions, in a
multicenter phase 2 trial on 23 patients with PCNSL relapsed or refractory
to HD-MTX (Reni et al., 2004). Considering it permeates the BBB, is well
tolerated even in elderly patients, and exhibits additive cytotoxic activity
with radiotherapy, temozolomide could be used as induction, maintenance,
or radiomimetic treatment against PCNSL.
Topotecan, a camptothecin derivative that inhibits enzyme topoisom-
erase I, has been tested in 16 patients with refractory or relapsed PCNSL,
obtaining four complete remissions and two partial remissions (overall
response rate: 38%) and a one-year progression-free survival rate of 13%
(Fischer et al., 2004). Promising results but on small groups of patients with
relapsed PCNSL have been reported by using ifosfamide and trofosfamide
(Jahnke et al., 2005), while infusional 5 bromo-2'-deoxyuridine given as
radiomimetic with whole brain radiotherapy has been associated with mod-
est disease control and unacceptable neurotoxicity (Dabaja et al., 2003).
Rituximab, a human-mouse chimeric anti-CD20 antibody active against
B-cell lymphomas, is an intriguing investigational drug. High doses of this
drug can be safely infused to attain higher cerebrospinal uid (CSF) con-
centrations ( 1.7% of serum level) (Raizer et al., 2000). However, anec-
dotal experience with intravenous rituximab shows disappointing results
(Harjunpaa et al., 2001). Conversely, promising results and excellent toler-
ance were reported in some cases of leptomeningeal lymphoma treated with
intraventricular rituximab (Schulz et al., 2004). However, these patients
died early because of progression of intraparenchymal lesions (Schulz et
al., 2004), and duration of response of leptomeningeal disease remains to
be dened. Recently, rituximab was used in association with temozolomide
in seven patients with CNS lymphoma, obtaining ve complete remissions
and two partial responses, with a median response duration and survival
of six and eight months, respectively (Wong et al., 2004). This combina-
tion was well tolerated and active in elderly and heavily pretreated patients
(Wong et al., 2004). However, it is not possible to know if response was
due to one or both drugs.
New Strategies: Chemoradiotherapy is associated with a higher risk
of neurotoxicity in PCNSL patients. Thus, some authorities focused their
efforts on new strategies, that is, BBBD and high-dose chemotherapy sup-
ported by autologous peripheral-blood stem-cell transplantation (APBSCT),
to dose intensify chemotherapy and eliminate the need for consolidation
radiotherapy. BBBD by intra-arterial infusion of hypertonic manni-
tol followed by intra-arterial cytostatics delivery is a strategy leading to
increased drug concentrations in the lymphoma-inltrated brain to enhance
survival. BBBD plus HD-MTX has been associated with ve-year survival
of 42%, and a 14% cognitive loss rate at one year (Kraemer et al., 2002).
In relapsed patients, carboplatin-based chemotherapy plus BBBD produced
a 36% response rate, with a median duration of 6.8 months (Tyson et al.,
2003). Given its good efcacy and acceptable complication rates, the role
of BBBD deserves further investigation in PCNSL.
Preliminary results indicate that high-dose chemotherapy supported by
APBSCT is feasible in PCNSL patients. In one study on 28 patients with
newly diagnosed PCNSL (Abrey et al., 2001), HD-MTX and HD-cytarabine,
followed by BEAM consolidation chemotherapy and APBSCT was well
tolerated, but only ve remained in remission at a median of 26 months
after transplantation. In an ongoing study, 19 of 24 enrolled patients have
achieved a complete remission, without relevant toxicity, after a combi-
nation of MTX, thiotepa, and cytarabine, followed by high-dose BCNU
and thiotepa and hyperfractionated radiotherapy (Illerhaus et al., 2001).
In a study on 22 patients with recurrent or refractory primary CNS or
intraocular lymphoma, induction cytarabine and etoposide followed by
high-dose thiotepa, busulfan, and cyclophosphamide produced a complete
remission rate of 72%, with a three-year overall survival of 64%, but with
a signicant treatment-related morbidity/mortality in elderly individuals
and risk of neurotoxicity in pre-irradiated patients (Soussain et al., 2001).
The role of high-dose chemotherapy and APBSCT in PCNSL remains to be
dened, considering that worldwide experience is still limited, and further
studies will need to be done to identify the optimal induction and high-dose
chemotherapy regimens.
Primary central nervous system lymphomas inltrate the subarachnoid
space and eyes in a variable proportion of cases (Ferreri et al., 2002). These
areas are considered as sanctuaries for conventional chemotherapy. Even
if MTX doses greater than or equal to 3 g/m
2
lead to therapeutic concentra-
tions and eradication of neoplastic cells from CSF (Guha-Thakurta et al.,
1999; Shapiro et al., 1975), some authorities suggest adding intrathecal
chemotherapy for meningeal treatment, mostly in cases with positive CSF
cytology. Drugs (MTX, cytarabine, and steroids) are delivered by intra-
thecal or intraventricular (Ommayas reservoir) route, and, importantly,
a sustained release formulation of cytarabine (liposomal cytarabine) for
intrathecal injection is available and allows dosing once every 14 days
(Jaeckle et al., 2001). Indications and efcacy of intrathecal chemotherapy
are, however, debatable. In fact, this strategy is associated with increased
risks of neurotoxicity and chemical meningitis (Bessell et al., 2002; Ferreri
et al., 2002), and its efcacy in PCNSL patients has not been prospectively
assessed. Moreover, leptomeningeal relapse is almost always associated
with brain recurrence (Ferreri et al., 2002), which constitutes the cardinal
prognostic event in PCNSL, obscuring the effect of concurrent leptomenin-
geal relapse on survival and, consequently, the potential benet of intrathe-
cal chemotherapy.
Chemotherapy efcacy against intraocular lymphoma is dependent
on intraocular pharmacokinetics, which is not well understood. One case
series suggests that micromolar concentrations of MTX are achieved in
the aqueous and vitreous humor when the drug is given at a dose of 8 g/m
(Batchelor et al., 2003; Ferreri et al., 2002). However, intravitreal drug
concentration is erratic, it is not predictive of response, and it is lower in
the vitreous humor, where lymphomatous cells usually grow, with respect to
the aqueous humor (Batchelor et al., 2003). These difculties have induced
investigators to establish protocols using repeated intravitreal injections
of MTX, with or without thiotepa, which are associated with promising
results and reduced morbidity (Smith et al., 2002).
Perspectives: The optimum treatment of PCNSL remains a relevant
challenge for international cooperation (Ferreri et al., 2003b). Collabora-
tive efforts should be focused on the identication of new active drugs and
combinations and on the role of emerging strategies against NHL. Differ-
ent combinations of strategies may be needed because of the capability of
lymphomatous cells to inltrate more than one compartment of the CNS.
To improve our knowledge of the molecular mechanisms underlying genesis
and dissemination of malignant lymphocytes constitutes an essential step to
improve therapeutic efcacy, and the establishment of PCNSL animal mod-
els will allow us to investigate a variety of novel molecules to be included in
the armamentarium against PCNSL.
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232. EXTENDED ABSTRACT: STROKES IN CANCER
PATIENTS: CEREBRAL INFARCTION, CEREBRAL
HEMORRHAGE, DISSEMINATED INTRAVASCULAR
COAGULATION, NEOPLASMS
Lisa R. Rogers; Hermelin Brain Tumor Center, Henry Ford Hospital
Detroit, MI, USA
Stroke in the cancer patient is rarely associated with the common causes
of stroke in patients without cancer. The type of stroke is tumor-specic
and is often associated with the stage of cancer and type of antineoplastic
therapy (Cestari et al, 2004; Graus et al., 1985).
Nonmetastatic
1. Coagulopathy
Infarction/ Thrombosis: A hypercoagulable state frequently accom-
panies carcinomas due to a complex interplay of immature cancer blood
vessels, inammation, and interaction of the host blood vessels with proco-
agulant substances secreted by the tumor. In nonbacterial thrombotic endo-
carditis (NBTE), sterile platelet-brin vegetations develop on heart valves.
This material embolizes to the brain and is accompanied by cerebral intra-
vascular thrombosis due to the underlying hypercoagulable state. Other
cancer patients have disseminated intravascular coagulation (DIC) and
diffuse small-vessel cerebral thrombosis without NBTE. Hypercoagulabil-
ity, sometimes from chemotherapy administration, underlies venous sinus
thrombosis, typically in patients with leukemia and lymphoma (Raizer and
DeAngelis, 2000). Nonbacterial thrombotic endocarditis results in focal or
multifocal cerebral signs from TIA (transient ischemic attack) or infarction
(Rogers et al., 1987). Confusion alone or with focal signs or partial seizures
may result from disseminated thrombosis in NBTE or DIC. Venous occlu-
sion typically causes a headache that is accompanied by seizures or focal
signs if infarction or hemorrhage develops. Signs of systemic thrombosis
may be observed in NBTE and DIC, but laboratory tests of coagulation
function are often not diagnostic. Cardiac vegetations visualized on trans-
esophageal echocardiography and evidence of systemic arterial or venous
thrombosis are clues to NBTE. MRI in NBTE will typically reveal varying
sizes of infarctions in multiple territories (Singhal et al., 2002). MRI or
magnetic resonance venography (MRV) is diagnostic of venous occlusion.
Optimal therapy for cancer-related hypercoagulability is not known and
should be individualized. Heparin should be considered for NBTE and DIC.
Venous occlusion may require anticoagulation, thrombolysis, or thrombec-
tomy but more often can be observed.
Hemorrhage: Acute DIC is most common in leukemia, especially
myelogenous leukemias. In acute promyelocytic leukemia, procoagulants
released from the tumor activate the clotting pathway and deplete clotting
factors. In other cancer patients, cerebral hemorrhage results from throm-
bocytopenia that is due to marrow metastasis, marrow suppression from
radiation or chemotherapy, or microangiopathic hemolytic anemia or liver
dysfunction. Acute or subacute headache, focal signs, vomiting, and/or
encephalopathy are signs of parenchymal or subdural hemorrhage from
coagulopathy. There may also be systemic bleeding. In acute DIC there
may also be systemic thrombosis. Microangiopathic hemolytic anemia also
causes pulmonary edema, hypertension, and renal insufciency. CT or
MRI will show single or multiple parenchymal or subdural hemorrhages.
Low platelets and brinogen, elevated prothrombin time, activated partial
thromboplastin time, and D-dimer are signs of acute DIC. Treatment for
DIC is directed to the tumor and replacement of clotting factors. Sometimes
anticoagulation is indicated. Subdural hemorrhages from coagulopathy can
usually be managed conservatively (Graus et al., 1996).
2. Treatment-Related
Infarction/ Thrombosis: Arterial or venous thrombosis is an uncom-
mon complication of chemotherapy, possibly related to vasospasm, vascu-
litis, or effects on the coagulation system. It is most common in children
with leukemia who develop venous sinus thrombosis after induction ther-
apy with L-asparaginase. Arterial infarction is also reported with cisplatin
administration and in breast cancer patients receiving tamoxifen and multi-
agent chemotherapy (Bushnell and Goldstein, 2004). Therapeutic radiation
to treat head and neck cancer is associated with accelerated carotid ath-
erosclerosis (Dorresteijn et al., 2002). Venous thrombosis typically causes
headache. There are focal signs if infarction or hemorrhage ensues. Che-
motherapy and radiationrelated thrombosis may result in TIA or infarc-
tion. Long segments of carotid stenosis conned to the area of radiation are
visualized on angiography in radiation-induced atherosclerosis. Radiation-
induced carotid disease is effectively treated surgically.
Hemorrhage: Radiation or chemotherapy with marrow suppression
may result in thrombocytopenia and brain, subdural, or subarachnoid
hemorrhage. The hemolytic-uremic syndrome is a complication of some
chemotherapies, especially mitomycin (Gordon and Kwaan, 1999).
3. Other
Infarction: Fungal septic embolus is a rare cause of stroke, resulting
in symptomatic bland or hemorrhagic infarctions, most often in leukemia
patients after bone marrow transplantation. Granulomatous angiitis is a
rare complication of lymphoma or leukemia.
Metastatic
1. Vessel Compression/Inltration
Infarction/Thrombosis: Metastatic tumor in the skull or meninges can
produce thrombosis in an underlying venous sinus due to compression or
inltration. Parenchymal arterial compression or spasm is a rare compli-
cation of leptomeningeal metastasis. Intravascular lymphomatosis causes
infarction from proliferation of lymphoma cells within cerebral vessels.
Metastatic venous occlusion causes gradual signs of increased intracranial
pressure. Papilloedema is often present. Subacute and progressive focal signs
or encephalopathy result from lymphomatosis. MRI or MRV shows venous
occlusion and skull or dural enhancement in metastatic venous occlusion.
Infarction, enhancement, and nonspecic white matter changes are seen on
MRI in lymphomatosis (Williams et al., 1998). Treatment for neoplastic
vessel compression or inltration includes brain radiation therapy and/or
chemotherapy.
Hemorrhage: Brain or dural metastasis can result in acute or subacute
hemorrhage. The most common parenchymal tumors are lung cancer, mela-
noma, germ cell tumors, and thyroid or hepatocellular carcinoma. Dural
metastasis is most common in breast and prostate carcinoma, less frequent
in leukemia and lymphoma. Hyperleukocytosis in acute leukemia with
leukostasis and brain hemorrhage is now rare. Signs of parenchymal hem-
orrhage are typically a sudden headache and focal signs. Subdural hemor-
rhages present subacutely with headache and focal signs or encephalopathy.
A clue to neoplastic parenchymal hemorrhage on MRI is early edema and
enhancement, heterogeneous signal, and other brain areas of enhancement.
Metastatic subdural hemorrhages are accompanied by dural and/or skull
enhancement. A dural biopsy may be required for diagnosis. Steroids are
often indicated to treat tumor-associated edema. Removal of the parenchy-
mal hematoma or drainage of subdural uid, followed by brain radiation,
is indicated if the patient is symptomatic.
2. Embolism
Infarction: Cerebral TIA or infarction results from mucin or large
tumor emboli, typically from the lung (ONeill et al., 1987), less often
from cardiac or aortic arch tumors. Embolization may occur from surgi-
cal manipulation of the lung to remove cancer. Brain CT or MRI shows
focal or multifocal infarctions and may show enhancement from growing
tumor. Echocardiography is diagnostic of cardiac tumor. The treatment for
tumor embolus is brain radiation and treating the systemic tumor in order
to prevent further episodes.
Hemorrhage: Neoplastic aneurysms with brain hemorrhage develop
from tumor embolic material that results in vessel invasion and rupture.
This rare disorder is usually described in choriocarcinoma or lung carci-
noma (Murata et al., 1993). Angiography can be diagnostic but is normal if
the aneurysm is obliterated by the hemorrhage. Treatment is chemotherapy
for the systemic tumor and brain radiation.
References
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tamoxifen treatment for breast cancer. Neurology 63, 12301233.
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lis, L.M. (2004) Stroke in patients with cancer. Neurology 62, 2025
2030.
Dorresteijn, L.D., Kappelle, A.C., Boogerd, W., Klokman, W.J., Balm, A.J.,
Keus, R.B., van Leeuwen, F.E., and Bartelink, H. (2002) Increased risk
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than 60 years. J. Clin. Oncol. 20, 282288.
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tions in patients with cancer. Medicine 64, 1635.
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E., and Rosman, C. (1996) Neurologic complications of autologous and
allogeneic bone marrow transplantation in patients with leukemia: A
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233. EXTENDED ABSTRACT: PARANEOPLASTIC
NEUROLOGICAL SYNDROMES
F. Graus; Service of Neurology, Hospital Clinic, Barcelona, Spain
Paraneoplastic neurological syndromes (PNS) are important in clinical
practice because they are associated with specic types of tumors and usu-
ally antedate the diagnosis of the cancer that usually is in a localized stage
where the chances to cure the tumor are highest. The clinical evaluation
of patients with suspected PNS is difcult because similar syndromes may
occur in the absence of cancer and the tumor is not evident at the onset of
the neurological disorder in the majority of patients. Some neurological
syndromes dened as classical must suggest a paraneoplastic etiology as one
of the leading diagnoses (Table 1). Most PNS have in common the subacute
onset, severe neurological deterioration, and, in those involving the central
nervous system, frequent evidence of mild cerebrospinal uid pleocytosis or
IgG oligoclonal bands. The most helpful test that suggests the paraneoplas-
tic etiology of the syndrome is the detection of onconeural antibodies (Table
2). Recently, the term well-characterized onconeural antibody has been
introduced to designate those onconeural antibodies (Hu, Yo, Ri, Ma2,
CV2, and amphiphysin) for which (1) there are recognizable patterns on
routine immunohistochemistry and for which immunoblotting on recom-
binant proteins is available to conrm their specicities, (2) the number
of cases reported is associated with tumors, (3) the description of well-
characterized neurological syndromes is associated with the antibodies, (4)
the unambiguous identication of the antibodies occurs among different
studies, and (5) the frequency of these antibodies is less in patients without
cancer. Most onconeural antibodies are tightly associated with particular
PNS and tumor types. However, the predictive value depends on the onco-
neural antibody and the PNS.
Recently, a panel of neurologists interested in PNS suggested that there
should be two levels of diagnostic evidence to dene a neurological syn-
drome as paraneoplastic: denite and possible. Each level can be reached
combining a set of criteria (Fig. 1). The panel recognized that the term
possible may include true PNS but also the coincidental association of
two unrelated disorders (the neurological syndrome and cancer). However,
this level of evidence may be useful to identify disorders that in the future
may be upgraded to denite PNS and to recognize PNS based on the iden-
tication of specic trends such as a higher than expected association with
a specic type of cancer. The panel emphasized that denite and possible
PNS have in common the need to exclude other known causes that could
explain the neurological syndrome under study even if onconeural antibod-
ies are positive.
Early diagnosis of the underlying tumor affords the best chance to cure
the neoplasm. In addition, effective treatment of the neoplasm contributes
to improving or stabilizing the PNS. Early tumor diagnosis requires a high
index of suspicion by the radiologist who performs the radiological exami-
nation. Recently, positron emission tomography showed a better sensitivity
than thorax CT to demonstrate the underlying neoplasm. Sometimes the
tumor discovered is not the one usually associated with the PNS or the
onconeural antibody. In this situation, the tumor may be responsible for
the PNS, or the patient may harbor another tumor that is responsible for the
PNS. A way to solve this dilemma is to determine if the tumor expresses the
antigen recognized by the onconeural antibody.
The clinical course of PNS is not always uniform. Spontaneous
improvement is reported in a few patients with several PNS. Furthermore,
some patients with PNS may present with a slowly indolent clinical course
over years in absence of any treatment.

Several immunosuppressor thera-
pies including corticosteroids, plasmapheresis, and intravenous high-dose
immunoglobulins have been used in the treatment of PNS. These therapies
are useful in the opsoclonus-myoclonus syndrome associated with neuro-
blastoma, with LEMS, with multineuritis with vasculitis, with dermato-
myositis, and in a few patients, with limbic encephalitis, particularly those
with anti-Ma2 antibodies. In most of these disorders, the damage to the
nervous system is functional more than structural, so a clinical improve-
ment may be expected after treatment.
In PNS with neuronal degeneration such as paraneoplastic cerebellar
degeneration, immunosuppressor therapies have been not successful. How-
ever, we favor a trial of immunosuppressor or immunomodulating drugs
based on the evidence that these PNS probably are immune mediated and on
occasional case reports that they improved with intravenous immunoglobu-
lins or other immunotherapies. Although theoretically, immunosuppression
could exacerbate tumor growth, we did not nd that these treatments were
an adverse prognostic factor for survival.
References
Darnell, R.B., and Posner, J.B. (2003) Paraneoplastic syndromes involving
the nervous system. N. Engl. J. Med. 349, 15431554.
Das, A., Hochberg, F.H., and McNelis, S. (1999) A review of the therapy of
paraneoplastic neurologic syndromes. J. Neurooncol. 41, 181194.
Neurological
Syndrome
Classical Nonclassical
Tumor
present
Tumor
present
Tumor
absent
Tumor
absent
Onconeural ab.
absent
Onconeural ab.
absent
Onconeural ab.
present
Well-
characterized
onconeural ab.
Partially
characterized
onconeural ab.
High risk
for cancer
Improvement
after cancer
therapy
or
Onconeural ab.
present
Definite Possible Definite Possible Possible Definite
Onconeural
ab.
absent or
present
Fig. 1. Diagnostic criteria for
paraneoplastic neurological
syndromes
Graus, F., Delattre, J.Y., Antoine, J.C., et al. (2004) Recommended diag-
nostic criteria for paraneoplastic neurological syndromes. J. Neurol.
Neurosurg. Psychiatry 75, 11351140.
234. TREATMENT OF HUMAN GLIOMA CELLS WITH
HUMAN ANTI-HUMAN TNF-RELATED APOPTOSIS-
INDUCING LIGAND (TRAIL/APO2L) RECEPTOR
MONOCLONAL ANTIBODIES
M. Nagane, K. Kobayashi, and Y. Shiokawa; Department of
Neurosurgery, Kyorin University School of Medicine, Mitaka, Japan
TNF-related apoptosis-inducing ligand (TRAIL/Apo2L), a member of
TNF family, induces apoptosis preferentially in human tumor cells but not
in normal cells, suggesting TRAIL, through its cognate death receptors DR4
or DR5, may serve in a potential therapeutic role for intractable malignant
gliomas. We applied complete human anti-human TRAIL receptor mono-
clonal antibodies (mAbs) to specically target one of TRAILs death recep-
tors in human glioma cells, which could reduce potential TRAIL-induced
toxicity in human. All mAbs were provided by the Kirin Brewing Co. Ltd,
Tokyo. Fourteen human glioma cell lines were treated with either a human
anti-DR4 mAb (clone B12), or anti-DR5 mAbs (clones E11 and H48), and
their cytotoxic effects were determined by using MTT assays. Anti-TRAIL
receptor mAb-induced cytotoxicity was compared with that induced by
soluble human TRAIL. Caspase activation was evaluated by Western blot
analyses using whole cell lysates prepared after mAb treatments. Anti-DR5
mAb treatments induced signicant cytotoxicity in a majority of human
glioma cell lines tested, which was blocked in the presence of DR5-Fc, a
TRAIL-neutralizing fusion protein composed of the extracellular domain
of DR5. Sensitivity to anti-DR5 mAb correlated with that to soluble TRAIL
in these cells, suggesting that the apoptosis signals triggered by ligation of
DR5 with human mAbs may be transduced similarly to that by soluble
TRAIL. In contrast, anti-DR4 mAb treatment was ineffective in most
human glioma cell lines except two, and only one cell line exhibited cross
sensitivity to both mAbs. Established TRAIL-resistant sublines, T98G.
TR and LNZ308.TR, showed lower response rates to the mAb treatments.
Anti-DR5 mAb treatment resulted in cleavage and activation of initiator
and executioner caspases, as well as cleavage of poly(ADP-ribose) poly-
merase, an intrinsic substrate of caspase 3. Furthermore, treatment with
anti-DR5 mAbs suppressed growth of subcutaneous xenografts derived
from LNZ308 cells in athymic mice. These results suggest that DR5 may
represent the major functional TRAIL receptor mediating receptor-induced
apoptosis in human glioma cells, and targeting DR5 with human mAb ago-
nistic to DR5 could provide a potential therapeutic strategy against intrac-
table malignant gliomas.
235. INHIBITION OF C-JUN N-TERMINAL KINASE
ENHANCES TEMOZOLOMIDE-INDUCED CYTOTOXICITY
IN HUMAN GLIOMA CELLS
Y. Hirose, S. Ohba, and T. Kawase; Neurosurgery, Keio University,
Tokyo, Japan
Previous studies revealed that the p38, a member of stress-activated
protein kinases (SAPKs), cooperates with the Chk1-pathway to bring about
TMZ-induced G2 arrest, and the inhibition of either pathway alone is suf-
cient to sensitize U87MG glioma cells to TMZ-induced cytotoxicity. We
hypothesized that other SAPKs might be involved in cellular responses to
DNA damage and that blocking of such protein might sensitize glioma cells
to chemotherapeutic agents. In the present study, we analyzed alteration of
c-Jun N-terminal kinase (JNK), another SAPK, in U87MG cells treated
with DNA-methylating agent temozolomide (TMZ). Immunoblot analysis
showed that JNK was phosphorylated 1 to 2 days after TMZ treatment.
Since a previous study suggested that TMZ induces severe DNA damage
1 to 2 days after the drug exposure through activation of DNA mismatch
repair system, we speculate that activation of JNK is triggered in response
to the creation of severe DNA damage, probably DNA double strand breaks
which are potentially lethal to the cells. To analyze the role of JNK phos-
phorylation in survival of glioma cells with DNA damage, we pre- (for
24 h) and post-(for 72 h) treated U87MG cells with JNK inhibitor (Cal-
biochem, USA) in combination with TMZ treatment. Colony formation
efciency assay revealed that the clonogenicity of TMZ-treated U87MG
cells was remarkably reduced by JNK inhibitor at 200 nM or higher concen-
tration. Immunoblot for phosphorylated cdc2 revealed that this potentia-
tion of TMZ-induced cytotoxicity was not associated with abrogation of
G2 checkpoint pathway. Phosphorylation of JNK target protein c-Jun was
inhibited with 200 nM JNK inhibitor. However, phosphorylation of ATF-2,
another JNK target, was not affected by this concentration of JNK inhibi-
tor, and it was suggested that c-Jun-related responses were more important
in JNK-mediated survival of glioma cells with DNA damage. Finally, we
performed similar experiments using another human glioma cell line, U251,
and conrmed that the events mentioned above were not cell line-specic.
The mechanism of the JNK inhibitor-induced enhancement of the cyto-
toxicity of TMZ is still unclear, and JNK inhibitors are not available for
clinical use. Nonetheless, further investigation based on the present data
may provide a viable approach for the sensitization of human gliomas to
TMZ-induced cytotoxicity.
236. BLOCKADE OF THE PI3-KINASE P110A CATALYTIC
SUBUNIT INDUCES G2M ARREST AND APOPTOSIS IN
HUMAN GLIOMA CELL LINES
W. Weiss,
1
Q.-W. Fan,
1
Z. Knight,
2
and K. Shokat
2
;
1
Neurology,
Pediatrics, and Neurological Surgery,
2
Cellular and Molecular
Pharmacology, University of California, San Francisco, California, USA
Amplication of EGFR occurs in 40% of astrocytomas and correlates
with advanced disease. Activation of phosphatidylinositol-3-kinase (PI3-
kinase) also occurs commonly in glioma and occurs in part through loss of
the tumor suppressor PTEN. Because the EGFR and PI3-kinases are acti-
vated in glioma and in other human cancers, combination therapies directed
against these kinases offer a mechanistic rationale to improve therapy. In
published work, we showed that inhibition of EGFR cooperated with inhi-
bition of PI3-kinase in the preclinical therapy of glioma. The PI3-kinases
constitute a complex protein family classied according to structure and
substrate specicities. Despite known differences in upstream activation,
the physiological roles of individual PI3-kinase isoforms and the contribu-
tions of individual isoforms to specic malignancies remains poorly under-
stood. The small-molecule PI3-kinase inhibitors LY294002 and wortman-
nin have been instrumental tools to dissect basic elements of PI3-kinase
signaling. As a consequence of indiscriminately inhibiting all PI3-kinases
and a large number of related proteins, LY294002 and wortmannin are too
toxic to be used in patients. Thus, the utility of small-molecule PI3-kinase
inhibitors in clinical practice requires development of new, more selective
inhibitors that can be safely and effectively used in patients. To address
the role of particular PI3-kinase isoforms in glioma, we have synthesized
12 isoform-selective inhibitors of particular PI3-kinase subunits likely to
contribute to glioma and have characterized the IC50 values against 20
recombinant kinase targets. These agents represent the rst new tools avail-
able in a decade for analysis of PI3-kinase signaling. We have screened all of
these compounds against a panel of astrocytoma cell lines. Although most
of these inhibitors were quite potent in blocking the PI3-kinase downstream
target Akt, only one inhibitor (selectivity: p110a DNA-Protein Kinase
p110g) induced signicant growth arrest and apoptosis in the entire panel
of human glioma cell lines and was most effective against cells wild-type for
PTEN. This study demonstrates that the PI3-kinase catalytic subunit p110a
plays an important role in proliferation and survival of glioma.
237. LOW-MOLECULAR-WEIGHT EGFR/KDR TYROSINE
KINASE INHIBITOR OFFERS COMBINATORIAL BENEFIT
WITH A RAPAMYCIN DERIVATIVE BASED ON PTEN
STATUS
J. Rich,
1
Q. Shi,
2
M. Hjelmeland,
2
R. Goudar,
1
S. Keir,
2
R. McLendon,
3

E. Reese,
2
P. Traxler,
4
H. Lane,
4
D. Bigner,
3
and H. Friedman
2
;
Departments of
1
Medicine,
2
Surgery, and
3
Pathology, Duke University
Medical Center, Durham, North Carolina, USA;
4
Novartis Institutes for
BioMedical Research Basel, Oncology, Basel, Switzerland
Malignant gliomas are highly lethal tumors that display striking genetic
heterogeneity. Novel therapies that inhibit a single molecular target may
slow tumor progression, but tumors are likely not dependent on a signal
transduction pathway. We recently reported the rst completed trial of
an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor,
getinib, in recurrent glioblastomas. Although a subset of patients experi-
enced long-term control of tumor growth, the majority of patients suffered
progression of their tumors. The molecular mechanisms by which cancers
develop resistance to EGFR inhibitors remains poorly understood. As EGFR
may have signicant impact on tumor growth through its pro-angiogenic
effects, independent vascular endothelial growth factor receptor 2 (kinase
domain region, KDR) activity may provide an important survival advan-
tage with the withdrawal of EGFR effects. Additionally, PTEN-decient
glioma cell lines display increased sensitivity to mammalian target of rapa-
mycin (mTOR) inhibition as compared with those with wild-type PTEN.
AEE788 is a novel orally active tyrosine kinase inhibitor that decreases the
kinase activity associated with EGFR and KDR. RAD001 [everolimus] is
an orally available mTOR inhibitor structurally related to rapamycin. We
hypothesized that combined inhibition of upstream EGFR and KDR recep-
tors with AEE788 and inhibition of the downstream mTOR pathway with
RAD001 would result in increased efcacy against gliomas compared to
single-agent therapy. In vitro experiments showed that the combination of
AEE788 and RAD001 resulted in increased rates of cell cycle arrest and
apoptosis, and reduced proliferation more than either agent alone. Com-
bined AEE788 and RAD001 administered orally to athymic mice bearing
established human malignant glioma tumor xenografts expressing mutant
Pten resulted in greater tumor growth inhibition and greater increases in
median survival than monotherapy. In contrast, a malignant glioma xeno-
graft expressing a wild-type Pten was more signicantly growth inhibited
upon AEE788 treatment as monotherapy but the addition of RAD001 had
only a marginal impact. These studies suggest that simultaneous inhibition
of growth factor receptor and mTOR pathways offer increased benet in
glioma therapy with preferential benet in tumors with disruption of Pten
function. This work was also supported by grants from Accelerate Brain
Cancer Cure, Pediatric Brain Tumor Foundation of the United States, and
NIH grant NS047409. J.N.R. is a Damon Runyon-Lilly Clinical Investiga-
tor and a Sidney Kimmel Cancer Foundation Scholar.
238. HUMAN, TUMOR-FOUNDING NEURAL STEM CELLS
AND TUMOR STEM CELL LINES FOR THE DIAGNOSIS AND
THERAPY OF GLIOBLASTOMAS
A. Vescovi,
3
R. Galli,
2
E. Binda,
2
S. Piccirillo,
2
G. Broggi,
1
and
F. DiMeco
1
;
1
Neurosurgery, Istituto Nazionale Neurologico, C. Besta,
Milan;
2
Stem Cell Research Institute, Fondazione San Raffaele, Milan;
3
University of Milan-Bicocca, Milan; Italy
We have recently provided the initial evidence that, unlike other brain
malignancies, the lethal glioblastoma multiforme (GBM) contains neural
precursors endowed with all of the critical features expected from NSCs.
Similar, yet not identical, to their normal NSC counterpart, these precur-
sors emerge as unipotent (astroglial) in vivo and multipotent (neuronal-
astroglial-oligodendroglial) in culture. More importantly, these cells can
act as tumor-founding cells down to the clonal level and can establish
tumors which closely resemble the main histological, cytological, and
architectural features of the human disease, even when challenged through
serial transplantation. Thus, cells possessing all the characteristics expected
from tumor neural stem cells from GBMs, including the typical inltrating
and migratory capacity expected from malignant glioma cells, appear to
be involved in the growth and recurrence of adult human GBMs. Such fea-
tures have never been observed previously in the use of common xenograft
or allograft-based brain tumor models. Our report also describes tumor
neural stem cells (TNSCs) that can be used to routinely establish single
patient-derived GBM cell lines in a quick and reproducible fashion. Impor-
tantly, TNSCs from different patients retain their distinctive, line-specic
proliferation and differentiation attributes which appear to be genetically
determined, as shown by the establishment of clonal TNSCs, which possess
stable properties identical to those of their parental bulk cultures. TNSCs
remain unaltered after multiple in vitro passages and even serial in vivo
orthotopic transplantation. Using these lines we are now exploring the pos-
sibility that the same key genetic, epigenetic, and extracellular cues that are
involved in the maintenance of stem cells and their fate regulation may also
be at work in TNSCs so as to prove that the body of knowledge that has
emerged from studying basic brain stem cell physiology may be harnessed
to identify new therapeutic targets and approaches in neuro-oncology. We
shall present recent ndings which show that some cues act upon neural
stem cells. In our hands, TNSCs also provide an invaluable tool for the in
vivo modeling and studying of GBMs, particularly in view of their patient-
specic features. As a result, these cells may provide the means to improve
diagnosis and develop patient-tailored therapies.
239. A BISPECIFIC IMMUNOTOXIN (DTAT13) TARGETING
HUMAN INTERLEUKIN-13 AND UROKINASE-TYPE
PLASMINOGEN ACTIVATOR RECEPTORS IN A MOUSE
XENOGRAFT MODEL
W.A. Hall,
1,3
E. Rustamzadeh,
1
D.A. Vallera,
2
D.A. Todhunter,
3
and
D.A. Vallera
3
;
1
Neurosurgery;
2
Therapeutic Radiology;
3
Cancer Center,
University of Minnesota, Minneapolis, Minnesota, USA
A bispecic immunotoxin (IT) DTAT13 was synthesized in order to
target simultaneously the urokinase-type plasminogen activator receptor
(uPAR)-expressing tumor neovasculature and IL-13 receptor expressing
glioblastoma cells with the goal of intratumoral administration for brain
tumors. The recombinant hybrid was created by using the non-internalizing
N-terminal fragment of uPA (ATF) and the IL-13 molecule for binding plus
the catalytic and translocation portion of diphtheria toxin (DT) for killing.
The 71 kDa protein was highly selective for human glioblastoma in vitro
showing no loss on binding compared with DTAT and DTIL13 controls. In
vivo, DTAT13 caused the regression of small tumors when administered at
10 g/day given on a ve-dose schedule every other day. DTAT13 was able
to target both overexpressed uPAR and the vasculature, as demonstrated
by its ability to kill HUVEC cells. Also, mortality studies indicated that
DTAT13 was less toxic than DTAT or DTAT13. These ndings indicate
that bispecic IT may allow treatment of a broader subset of antigenically
diverse patients while simultaneously reducing the exposure to toxin that is
required if two separate agents were employed.
240. GENOME-WIDE ALLELIC IMBALANCE ANALYSIS
OF PEDIATRIC GLIOMAS BY HIGH-DENSITY SINGLE
NUCLEOTIDE POLYMORPHIC ALLELE (SNP) ARRAY
K. Wong, Y.T.M. Tsang, Y. Chang, and C.C. Lau; Pediatric/Hematology-
Oncology, Texas Childrens Cancer Center, Baylor College of Medicine,
Houston, Texas, USA
In the Childrens Cancer Group high-grade glioma study CCG-945,
out of 250 high-grade glioma cases diagnosed by local pathologists, 70
cases were reclassied as low-grade glioma after central consensus pathol-
ogy review. This indicates a need for additional criteria other than mor-
phology, such as genome-wide genotyping (allelic imbalance analysis), to
improve the accuracy of histopathologic classication for these tumors.
More importantly, the etiology and molecular pathogenesis of pediatric
gliomas remain unclear. Brain tumor tissues were obtained under an IRB-
approved protocol after informed consents were obtained from patients
undergoing tumor resection at Texas Childrens Hospital, Baylor College
of Medicine. Portions of the tumors were xed in 10% formaldehyde and
embedded in parafn for sectioning and pathological diagnosis, and the
residual tissues were snap-frozen in liquid nitrogen and stored at 80C
for DNA extraction. All tumor tissues were obtained at initial diagnosis
with no prior exposure to chemotherapy

or radiation. Totally, 7 low-grade
gliomas (5 JPA, 1 ganglioglioma, 1 astrocytoma) and 9 high-grade glio-
mas (GBM) were analyzed by SNP array that contains 11,560 SNP alleles
spanning the human genome with a median intermarker distance of 105
kb. Sixteen pediatric gliomas were analyzed by SNP arrays. No loss of het-
erozygosity (LOH) was detected in any of the of the 11,562 SNP loci for
the ve JPA tissues studied. The ganglioglioma has LOH in 7 SNP loci on
chromosome 9p while the astrocytoma has LOH in 28 SNP loci on chromo-
some 6q. On the other hand, high-grade gliomas are very heterogeneous in
that the number of SNP loci with LOH varied from 52 to 2125. Signicant
LOH cytoband regions in GBM include 4q, 6q, 9p, 12, 13q, 14q, 17, 18p,
and 19q. We also detected amplication of SNP loci near the genes EGFR
and PDGFRA in two different cases of GBM. No observable allelic imbal-
ance was detected in JPA, and allelic imbalance in other low-grade gliomas
only involves a single chromosome. On the other hand, allelic imbalance
in high-grade gliomas is quite variable and involves multiple chromosomes.
The simultaneous measurement of DNA copy number changes and LOH
by SNP arrays should enhance our ability to discover cancer-causing genes
and to rene the diagnosis of pediatric gliomas.
241. DISSECTING INTRATUMORAL HETEROGENEITY IN
UNTREATED GLIOBLASTOMA: TALES FROM THE EDGE
T. Van Meter,
1
C. Dumur,
2
N. Hafez,
2
C. Garrett,
2
H. Fillmore,
1
and
W. Broaddus
1
;
1
Neurosurgery;
2
Pathology, Virginia Commonwealth
University, Richmond, Virginia, USA
Glioblastoma multiforme is the most aggressive and treatment-resistant
adult primary brain tumor. The molecular changes that underlie tumor het-
erogeneity in glioblastoma have not been studied in detail. We have under-
taken a detailed analysis of tissue prospectively collected intra-operatively
using Stealth imaging-assisted tissue extraction. This method was used to
isolate tissue samples from multiple intra-tumoral regions in untreated glio-
blastoma, corresponding to the enhancing tumor rim and areas of hypoxic
tumor core. Affymetrix HG-U133A high-density oligonucleotide arrays
were used to assess differences in gene expression proles in the different
regions. Our approach used an RNA extraction protocol paired with in-
process histological scoring of tissue samples using H&E staining of frozen
sections. Tumor gene expression proles from different tumor regions were
compared and correlated with percent tumor, percent necrosis, and other
histological features. In this report, we focused on genes upregulated in the
enhancing periphery of regions bearing 90% tumor versus normal brain,
compared to the core regions. We have analyzed the resulting normalized
data sets using a series of 3 algorithms (MBEI, MAS5 and RMA) to provide
a consensus prole of transcriptomic differences between periphery and
core samples. Previously, we have reported that EGFR and AKT signaling
are upregulated in the tumor periphery. Here we report that tumor cells in
the periphery of untreated GBMs overexpress additional survival factors
in the TNF superfamily, putative regulators of MAP kinase signaling, and
several previously unreported transcriptional regulators including LHX2,
FoxF1, Sox11, and Shox. Upregulation of EGFR and AKT signaling seen in
the tumor periphery, and the putative survival pathways reported here, are
consistent with the notion of apoptotic suppression in cells of the invasive
rim in GBM. Novel mechanisms of upregulated survival signaling may be
important to the inltrative phenotype and treatment resistance in GBM.
242. CHROMOSOMAL TILE PATH ARRAY- CGH ANALYSIS
IN ASTROCYTIC TUMORS LEADS TO IDENTIFICATION
OF A NOVEL CANDIDATE TUMOR SUPPRESSOR GENE ON
CHROMOSOME 22
K. Ichimura, T.J. Seng, D.M. Pearson, L. Liu, and V.P. Collins;
Molecular Histopathology, University of Cambridge, Cambridge, UK
Adult astrocytic tumors have complex and diverse genotypes. A number
of oncogenes and tumor suppressor genes (TSGs) have been implicated in
the development and/or progression of these tumors, including CDKN2A/
CDKN2B/p14ARF, RB1, CDK4/ CDK6, MDM2, TP53, EGFR, and
PTEN. In addition, numerous studies of microsatellite analysis and meta-
phase CGH have revealed frequent losses, gains, and amplications in many
chromosomal regions in astrocytic tumors, suggesting the presence of as yet
identied novel TSGs and oncogenes. One such chromosome is chromo-
some 22. However, those conventional techniques have failed to narrow
down the critical regions and to pin-point the target genes because of low
resolution and/or lack of correlation to sequence. We have constructed a
chromosome 22 tile path microarray for CGH (array-CGH) in order to pre-
cisely investigate the chromosomal 22 abnormalities of astrocytic tumors.
The technique has a number of advantages in that (1) it does not rely on
naturally occurring polymorphisms, (2) the ndings can be directly linked
to published human genome sequences, and (3) it allows quantitative assess-
ment of copy number at each clone. A tile path clone set for chromosome
22, which covers 82% of 22q with 443 BAC/PAC/cosmid/fosmid clones,
has been obtained from the Wellcome Trust Sanger Institute. The array was
constructed according to the published protocol using modied DOP-PCR
method. A total of 126 astrocytic tumors consisting of 92 glioblastomas
(GB), 29 anaplastic astrocytomas (AA), and 5 diffuse astrocytomas (A)
were subjected to the study. These tumors have also been examined for
allelic status using 28 microsatellite markers distributed along the entire
22q. Approval for the study has been obtained from the local ethical com-
mittee. The results showed good concordance between microsatellite and
array-CGH data. As a result of combined analysis using chromosome 22
array and microsatellite analysis, we identied 22q abnormalities in 38% of
GB, 33% of AA and 5% of A. Among several candidate regions identied,
we further investigated two overlapping homozygous deletions on 22q12.3
that spanned three clones. The region harbored three genes, YWHAH,
C22ORF24, and DEPDC5. Homozygous deletions were conrmed by
Southern hybridization and multiplex PCR. Gene-by-gene mutation analy-
sis revealed 3 different protein truncating mutations in 3 glioblastoma cell
lines and another somatic nonsense mutation in a primary glioblastoma and
its xenograft in DEPDC5. We have thus demonstrated that chromosomal
22 tile path array can accurately discriminate single copy number change
at each clone and that it is a powerful tool to identify critical regions. Tile
path arrays for chromosome 6, 7, and 10, as well as an array that covers the
whole genome with less than 1Mb interval, have also successfully been con-
structed and are being used for further investigation of astrocytic gliomas.
243. MOLECULAR SIGNATURES REFLECTING
DIFFERENTIATION STATUS OF HIGH- GRADE
ASTROCYTOMA IDENTIFY PROGNOSTIC SUBCLASSES
OF TUMOR AND DELINEATE A PATTERN OF DISEASE
PROGRESSION
H.S. Phillips,
1
S. Kharbanda,
1
R.H. Soriano,
1
W.F. Forrest,
1
T.D. Wu,
1

J. Nigro,
1
P.M. Willams,
1
L. Soroceanu,
1
A. Misra,
3
B.G. Feuerstein,
3

and K.D. Aldape
2
;
1
Genentech, Inc., San Francisco, California;
2
M.D.
Anderson Cancer Center, Houston, Texas;
3
University of California San
Francisco, San Francisco, California; USA
Gene expression proling of a set of 76 newly diagnosed high-grade
astrocytomas reveals molecular subtypes of tumor that differ in both sur-
vival times and in the subsets of genes whose expression is most strongly
associated with survival. Based on relative expression of 35 marker genes,
tumors are segregated into 3 groups, each of which has an expression signa-
ture characteristic of a distinct set of tissues. One tumor subclass displays a
median survival time (175 weeks) that is substantially longer than that of the
other subtypes (61 weeks and 71 weeks) and is distinguished by a marker
signature that resembles that of fetal brain or undifferentiated neural stem
cell lines. Markers of this tumor subtype include genes implicated in neu-
rogenesis and, among this tumor subset, one of the genes most strongly
correlated with survival is Numb, a Notch pathway antagonist critical for
regulation of forebrain neurogenesis. Tumors of the subclasses with poorer
prognoses display signatures dominated by features characteristic of either
proliferating tissues or tissues of mesenchymal origin. A Cox proportional
hazards model suggests that many genes strongly associated with survival
within one tumor subgroup do not exhibit equivalent effects across all
tumor groups. Utilizing matched primary-recurrent pairs of tumor samples
to evaluate changes in molecular signatures that accompany recurrence fol-
lowing surgery and radiation therapy, we nd that some tumors show sub-
type switching, most notably into the mesenchymal phenotype. Compara-
tive genomic hybridization analysis on a subset of samples reveals a strong
association between loss of chromosome 10, the most commonly occurring
aberration in GBM, and the mesenchymal signature. These ndings suggest
that aggressiveness of high-grade astrocytomas is governed in large part by
genetic and epigenetic changes that determine whether the tumor displays
neural lineage markers or adopts a phenotype indicative of an alternate dif-
ferentiation state. This work was supported in part by the National Brain
Tumor Foundation and NIH grants CA85799 and NS42927.
244. PIK3CA IS MUTATED IN OLIGODENDROGLIOMAS,
ASTROCYTOMAS, AND MEDULLOBLASTOMAS AND
ASSOCIATED WITH CHROMOSOME 1p, 19q LOH IN
OLIGODENDROGLIOMAS
D.C. Adamson,
1
D.K. Broderick,
1
C. Di,
1
Y.R. Samuels,
2
R.E.
McLendon,
1
D.W. Fults,
3
V.E. Velculescu,
2
A. Rasheed,
1
D.D. Bigner,
1

and H. Yan
1
;
1
Pediatric Brain Tumor Foundation Institute, Duke
University Medical Center, Durham, North Carolina;
2
Johns Hopkins
University Medical Institutions, Baltimore, Maryland ;
3
University of
Utah, Salt Lake City, Utah; USA
The phosphatidylinositol 3-kinase (PI3K) pathway helps to maintain a
delicate balance between cell survival and death and is interrupted by inac-
tivating mutations of PTEN, a PI3K phosphatase, in some cancers, includ-
ing glioblastomas. Mutations in PIK3CA, a member of the PI3K family,
have also been identied in glioblastomas, colorectal cancers, and gastric
cancers, and in a smaller fraction of breast and lung cancers. The 1p and
19q LOH in oligodendrogliomas is a well-established genetic phenomenon
which has important prognostic and therapeutic implications for patients
with these tumors. Evidence from several retrospective studies suggests
that allelic losses of 1p and 19q serve as molecular markers for response to
chemotherapy and radiation therapy and are an indicator of prolonged sur-
vival in patients with oligodendroglioma. We explored the role of PIK3CA
and PTEN mutations in brain tumors by sequencing it in a panel of 332
tumors in 7 different histological categories, as described by two board-
certied neuropathologists. We also performed a microsatellite analysis on
44 tumors with a panel of 6 markers for 1p and 19q LOH. A total of 19
PIK3CA mutations were identied in 17% of the anaplastic oligodendro-
gliomas (6/36), 6% of the oligodendrogliomas (2/32), 5% of the glioblas-
toma multiforme (5/105), 7% of the anaplastic astrocytomas (1/31), and
6% of the medulloblastomas (5/78). No mutations were seen in low-grade
astrocytomas or ependymomas. Furthermore, no PTEN mutations were
identied in tumors with PIK3CA mutations. We also found 1p and 19q
LOH in 60% (13/22) of the well-differentiated oligodendrogliomas and
81% (18/22) of the anaplastic oligodendrogliomas. The eight discovered
unique missense mutations in oligodendrogliomas clustered in the helical
and catalytic domains. Of note, all of the tumors with PIK3CA mutations
possessed the LOH of 1p and 19q. These observations demonstrate that
PIK3CA and PTEN mutations occur in a mutually exclusive manner in
brain tumors. This novel discovery of relatively common PIK3CA muta-
tions in anaplastic oligodendroglioma and the clear association with 1q,
19p LOH gives new insight into the molecular pathogenesis of these tumors
and suggests the need to evaluate outcomes of patients with PIK3CA muta-
tions. PIK3CA mutations may serve as important prognostic markers for
brain tumors with anaplastic behavior.
245. MOLECULAR CLASSIFICATION OF
OLIGODENDROGLIOMA IDENTIFIES TRANSCRIPTS
ASSOCIATED WITH RESPONSE TO CHEMOTHERAPY
P. French,
1
M. Kouwenhoven,
2
S. Swagemakers,
3
P. van der Spek,
3

and T. Luider,
1
J. Kros,
2
M. van den Bent,
1
and P. Sillevis Smitt
1
;
1
Departments of Neurology,
2
Pathology, and
3
Bioinformatics, Erasmus
MC, Rotterdam, The Netherlands
Malignant gliomas are the most common primary central nervous sys-
tem tumor in adults. One glioma subtype, the oligodendrogliomas, have
a markedly better prognosis than most others gliomas (median survival
is 510 years). A common genomic aberration in oligodendrogliomas is a
combined loss of the 1p and 19q chromosomal arms. Loss of these arms
is signicantly correlated with response to treatment (combined radiation
therapy and chemotherapy). In order to better understand the molecular
mechanisms that underlie chemosensitivity and potentially identify molecu-
lar pathways affected in oligodendrogliomas, we compared mRNA expres-
sion proles of 28 oligodendrogliomas and 6 control brains using Affyme-
trix HU133-plus 2 microarrays. We rst performed unsupervised clustering
to group samples on their similarities in mRNA expression prole. Three
subgroups can clearly be distinguished. Subgroup 1 consists of low-grade
tumors and control brains; subgroup 2 consists mainly of chemosensitive
tumors with loss of 1p, while subgroup 3 consists of chemoresistant tumors
that have retained both copies of 1p. Unsupervised clustering therefore read-
ily identies subgroups that are associated with tumor grade and prognosis.
We next performed supervised clustering to identify genes associated with
loss of the short arm of chromosome 1. Both FISH and LOH-PCR were
used to determine loss of 1p and 19q in our samples. Supervised clustering
identied 95 probesets as being differentially expressed between tumors
that have lost one copy of 1p compared to those that have retained both cop-
ies. Interestingly, 81/95 (85%) of these probesets are located either on chro-
mosome 1p or on 19q, and the average difference in expression level is 0.55
0.14. As allelic loss of these genes virtually halves their expression level,
it can be hypothesized that these genes have an allele-number-dependent
expression level. These genes can therefore serve as markers to identify
chromosomal aberrations. Finally, supervised clustering was performed to
identify genes associated with chemotherapeutic response. We identied
16 probesets that are signicantly differentially expressed between chemo-
sensitive and chemoresistant tumors. These genes can provide a prognostic
value as to whether the tumor will respond favorably to chemotherapy and
may identify molecular mechanisms that underlie chemosensitivity.
246. MEASURING GROWTH RATES OF LOW- GRADE
GLIOMAS MAY PREDICT EARLY MALIGNANT
TRANSFORMATION
J. Rees, H. Watt, C. Benton, N. Fox, P. Tofts, H. Jager, and A. Waldman;
Molecular Neuroscience, Institute of Neurology, UCL, London, UK
A majority of adult low-grade gliomas (LGGs) grow slowly for many
years and then, unpredictably, undergo malignant transformation. The
clinical management of LGGs is controversial as there is no proven benet
to intervention prior to transformation. We have developed a reproduc-
ible and sensitive method of measuring tumor volumes on MRI scans in
order to model growth rates of untreated adult LGGs and to determine
whether acceleration of tumor growth occurs prior to transformation. Cor-
onal-Oblique FLAIR and thin section Gd T1w sequences were obtained
six monthly from 33 patients with untreated LGGs who were recruited
into a multimodality imaging study. Tumor volumes were measured on
5-mm-thick sections with an interslice thickness of 1.5 mm from FLAIR
images with a semi-automatic intensity gradient-based thresholding pro-
gram with manual editing used when required. The tumor was contoured
on all covering slices, the contoured areas were saved as a region le, and
their combined volume was calculated. Percentage tumor growth rates per
year were derived from hierarchical regression modeling and compared
between different tumor histologies and between non-transformers (NT)
and transformers (T). Transformation was dened as clinical deterioration,
or the appearance of new or increased enhancement on the Gd studies, and
conrmed by biopsy of the enhancing region or resection of the tumor. Sev-
enteen patients transformed and 16 remained clinically and radiologically
stable (NT). The average annual growth rate in the NT group was 13%
(95% CI, 9%18%) and in the T group was 26% (95% CI, 21%32%)
up to six months prior to the transformation scan. This equates to a 12%
higher growth rate in the T group (95% CI, 5%18%; P 0.0001). Within
the T group, the average growth rate increased in the nal six-month trans-
forming period to 57% per annum (95% CI, 19%100%; P 0.08 com-
pared with earlier growth rates). Non-transforming astrocytomas grew at
the same rate as oligodendrogliomas (14%15% per year). We conclude
that LGG which subsequently transform grow signicantly faster prior to
malignant transformation than non-transforming tumors. There is no dif-
ference between growth rates of astrocytomas compared with oligodendro-
gliomas. Accurate measurement of tumor growth may offer an early marker
of malignant transformation and enable intervention before symptomatic
or radiological progression.
247. THE USE OF DIFFUSION TENSOR IMAGING IN
SURGICAL PLANNING FOR RESECTION OF LEFT FRONTAL
LOBE TUMORS
A. Beaumont,
1
H. Krouwer,
2
J. Ulmer,
3
and W. Mueller
1
; Departments
of
1
Neurosurgery,
2
Neurology, and
3
Radiology, Medical College of
Wisconsin, Milwaukee, Wisconsin, USA
The goal of surgical intervention for primary and metastatic intracere-
bral tumors is to maximize resection and minimize post-operative decit.
Techniques including awake surgery, electrocorticography, and functional
mapping help delineate functionally important cortical tissue prior to resec-
tion. Diffusion Tensor Imaging (DTI) is an MR technique that can dem-
onstrate white matter pathways. This study explores the use of DTI and
white matter tractography in surgical planning for resection of posterior left
frontal lobe tumors. Thirty-two cases of posterior left frontal lobe tumor
resection performed by one surgeon (WM) were retrospectively analyzed.
Fourteen patients had pre-operative DTI in addition to standard surgical
planning and resection techniques. DTI was performed by using established
protocols with post-processing to determine white matter pathways. Short-
and long-term post-operative neurological decits were recorded. Statisti-
cal signicance was assessed by using the Fisher exact test. The median
age of patients with and without DTI was 43.7 years (range, 28.856.8)
and 42.0 years (range, 19.390.4), respectively. There were no signicant
differences in sex distribution (18 males, 14 females) or median time of fol-
low-up (5.4 months; range, 0.427.7) in the two groups. Histopathological
diagnosis was similar in the two groups (40.6% grade II, 15.6% grade
III, 31.2% grade IV, and 12.5% metastatic/other). In the DTI group, 7
patients (50.0%) had no new neurological decits, and 7 (50.0%) developed
post-operative speech or motor decit. In the non-DTI group, 10 patients
(55.6%) had no new post-operative decits, and 8 (44.4%) had speech or
motor decits. New decits were transient and completely resolved in 5
(83.3%) patients with pre-operative DTI and in 1 (12.5%) patient with-
out DTI (P 0.025). One DTI patient had persistent speech and motor
decit, and one patient had rapid tumor recurrence with clinical decline
during rehabilitation for mild weakness and aphasia. In this study DTI
patients had a trend for higher rate of post-operative decit but with a high
rate of recovery. Decits in the non-DTI group were more permanent (P
0.025). This is consistent with DTI permitting more aggressive resection
without causing prolonged decit. White matter tractography may be a
useful adjunct in planning the extent of surgical resection for tumors in
functionally signicant areas.
248. MR IMAGING BASED BIOLOGICAL
CHARACTERIZATION OF NEWLY DIAGNOSED GBM
A. Pirzkall,
1
E. Ozturk,
2
R. Choy,
2
J. Lupo,
2
M. Lee,
2
S. Chang,
3
M.
Berger,
3
and S. Nelson
2
;
1
Radiation Oncology,
2
Center of Molecular and
Functional Imaging, and
3
Neurological Surgery, University of California
San Francisco, San Francisco, California, USA
The objective of this study was to characterize metabolic and physi-
ologic properties of newly diagnosed GBM with respect to its morphologic
properties derived from MRI. Fourteen patients with newly diagnosed
GBM, prior to any intervention, were studied with MRI and multivoxel
3D Proton magnetic resonance spectroscopy imaging (MRSI), perfusion-
(PWI) and diffusion-weighted imaging (DWI) to assess morphologic, meta-
bolic, and physiologic properties of the tumors studied. The MRI proto-
col included axial T2-weighted (FLAIR) and post-contrast T1-weighted
(SPGR) sequences. Three-dimensional MRSI was acquired with lactate
editing and a 1-cc nominal spatial resolution. Diffusion and perfusion data
were resampled to match the resolution of the MRSI allowing for a voxel-
by-voxel analysis. Evaluated parameters included choline (Cho), creatine (Cr),
N-acetyl aspartate (NAA), lactate (Lac) and lipid (Lip), and Cho- to-NAA
index (CNI) for MRSI; normalized (to normal appearing white matter)
Apparent Diffusion Coefcient (nADC) for DWI; and Peak Height (PH)
for PWI. Manually dened, mutually exclusive regions of interest (ROI)
included macroscopic necrosis (Necr), contrast enhancement (CEL) and T2
hyperintensity (T2L) lesion, and normal appearing white matter (NAWM).
CNI contours of 2 (CNI2), previously conrmed to correspond with
tumor inltration, were generated. A total of 3453 data voxels within the
MRSI selected volume were studied. All GBM exhibited CEL and T2L, and
macroscopic necrosis was present in 12/14 patients. A number of signicant
differences in metabolic and physiologic imaging characteristics were found
with respect to the dened anatomic and anatomic/metabolic ROIs. The
CEL in newly diagnosed GBM was found particularly heterogeneous and,
apart from Necr, exhibited the highest levels of Lac and Lip (within the
CNI4-inside-CEL) reecting micronecrosis and hypoxia, while simultane-
ously exhibiting signicantly increased perfusion PH which was highest in
CNI2,3-inside-CEL indicating increased cell division and neovasculature.
Most importantly, the CNI regions extending beyond the CEL, on the other
hand, exhibited clearly tumor suggestive metabolism with highest Cho,
increased PH, increased Lac, and to a lesser extent Lip, demarcating the
leading edge of the tumor. Regions of T2 hyperintensity had similar Cho,
decreased Cr and NAA, slightly increased Lac/Lip, much increased nADC,
and similar PH values compared to NAWM indicating a mixture of edema
and tumor cell inltration. Metabolic and physiologic imaging provides
additional insight into biological characteristics, extent, and heterogeneity
of brain gliomas likely enabling us to optimize our treatment planning both
for surgery and for radiation therapy and allowing us to improve patient
selection for respective treatment protocols.
249. HIGH SPECIFICITY AND SENSITIVITY OF
HIGH-RESOLUTION, BIPLANE DIGITAL SUBTRACTION
ANGIOGRAPHY IN THE DIAGNOSIS OF PATIENTS WITH
SUSPECTED GLIOBLASTOMA MULTIFORME: IMPACT ON
THE MANAGEMENT OF NONSURGICAL CASES
L. Mariani,
1
L. Schaller,
1
A. Fathi,
1
L. Remonda,
2
G. Schroth,
2
and R.
Seiler
1
;
1
Universittsklinik fr Neurochirurgie, Inselspital;
2
Institut fr
Pathologie, Universitt Bern, Switzerland
In a signicant subset of patients suspected to have supratentorial glio-
blastoma multiforme (GBM) based on MRI, biopsy does not have thera-
peutic consequences, is performed to rule out a hypothetic curable lesion, is
not always conclusive, and carries a signicant risk of bleeding and clinical
deterioration. Less invasive, but equally reliable diagnostic techniques are
warranted for these patients with a limited life expectancy. We routinely
perform preoperative digital subtraction angiography (DSA) in patients
suspected to have a GBM. We investigated the diagnostic specicity and
sensitivity of DSA in this patient population. Patients diagnosed between
1993 and 2002, with a supratentorial lesion compatible with a GBM based
on MRI, with a preoperative high-resolution, biplane DSA (1024 1024
matrix), and with a histological diagnosis were included. Neuroradiologists
blinded for the histological diagnosis analyzed the angiograms and assessed
the image quality and the presence of tumor-blush, pathological vessels
(PV), and an early venous drainage (EVD). They were asked to answer fol-
lowing questions about the lesion: Is it a tumor? Is it a malignant tumor?
Is it a GBM? One hundred eighty-six patients were eligible for the study
(32 stereotaxic biopsies, 154 surgical resections). No major complications
resulted from angiography. Preliminary data showed following results: (1)
The presence of PV associated with EVD at DSA was 100% specic for the
presence of a malignant tumor (77% a malignant glioma, 20% a metasta-
sis, 3% a PNET). (2) PV associated with EVD at DSA was present in 82%
of GBMs and 75% of all malignant tumors including GBM (sensitivity). We
conclude that in patients with supratentorial, intracerebral lesions compat-
ible with a GBM based on MRI, the presence of pathological vessels and of
an early venous drainage in high-resolution biplane DSA is both extremely
specic and highly sensitive for the diagnosis of a malignant tumor, mostly
a malignant glioma. In these patients, especially those in whom no therapy
is planned, the risk of biopsy can be avoided.
250. NEWLY DIAGNOSED GLIOBLASTOMA MULTIFORME:
PROTON MR SPECTROSCOPIC CHARACTERISTICS AND
CORRELATION WITH CLINICAL OUTCOME
J. Chang,
1
X. Li,
2
N. Butowski,
3
S. Chang,
3
M. Prados,
3
K. Lamborn,
3

M. Berger,
3
S. Nelson,
2
and S. Cha
1
; Departments of
1
Neuroradiology,
2
Radiology, and
3
Neurosurgery, University of California at San
Francisco, San Francisco, California, USA
Proton magnetic resonance spectroscopic imaging (1H MRSI) is a non-
invasive means of analyzing metabolic integrity within both normal and
diseased areas of the brain. The purpose of this study was to use 1H MRSI
to separately quantify levels of lactate (Lac), lipid (Lip) and choline to N-
acetyl aspartate ratio index (CNI) in two groups of glioblastoma multi-
forme (GBM) patients differentiated by time to progression (TTP) to assess
for relationships between clinical outcome and metabolic markers. Twelve
newly diagnosed GBM patients were recruited for this study. These patients
were divided into two groups based on clinical status: rapid progression
(RP, progression before 6 months; n 8) and moderate progression (MP,
progression after 6 months; n 4). In addition to pre-operative anatomic
MR imaging, all patients underwent 3D J-difference lactate-edited MRSI
using a PRESS volume selection technique. The lactate-edited method
required two acquisitions in each phase encoding step and provided reliable
separated quantications of Lip and Lac. Spectroscopic data was analyzed
for levels of Lip, Lac, and CNI. The metabolite levels were quantied by
using a software program developed in-house. The quantication was based
on a z-score, dened as the number of standard deviations (SD) a voxel
presented away from the control voxels, given a normal distribution with a
mean of 0 and a SD of 1. A z-score of 4 for Lac/Lip and 2 for CNI was
considered abnormal. The following measurement variables were derived
from each patients 1H MRSI exam: number of voxels with signicant z-
scores (sigZ), average z-score (avgZ), and Max z-score (maxZ). The average
volume of the T2L hyperintensite captured by the PRESS box was similar
among both groups. MRSI voxels contaminated with lipid artifact were
excluded from analysis. Results were as follows.
Group score derived
from an average of each # voxels avg Z max Z
patients preoperative
exam lac lip cni lac lip cni lac lip cni
Rapid Progression 26.3 21.8 28.9 6.8 10.6 3.8 11.8 20.1 6.3
Moderate Progression 19.0 8.0 21.0 6.4 7.8 4.4 10.3 13.1 8.4
Overall, the presence of Lac and that of CNI were comparable in the RP
and the MP groups. Lip, however, was found in higher quantities in the
RP group than in MP. This trend was observed under all measurement
variables, and was particularly notable under sigZ. Our preliminary data
suggests that lipid may predict clinical outcome in GBM patients and that
lipid is also a better predictor of outcome than Lac or CNI. The results of
this study suggest that metabolic information derived from 1H MRSI may
be a predictive marker of clinical outcome in patients with GBM. Further
study with larger sample size and longer follow-up will be conducted to
further evaluate 1H MRS variables in predicting clinical outcome.
253. ADVANCES IN MANAGEMENT OF EPENDYMOMA
R. Grundy; Childhood Brain Tumor Research Centre, University of
Nottingham, UK
Pediatric ependymomas are enigmatic tumors, and their clinical man-
agement remains one of the more difcult in pediatric oncology. There are
a number of controversies in ependymoma, and these re highlighted in my
address. Ependymomas are thought to arise from the ependymal lining of
the ventricles and lining of the central canal, though the cell of origin is
unknown. Indeed our understanding of the biology of this disease is still
limited. While a number of prognostic factors have been identied, these
are based on predominantly single-institution studies over long time periods
covering many different attitudes to both the diagnosis and treatment of
ependymoma. The most widely accepted prognostic factor is the degree of
surgical resection. However, 1/3 of the children with complete resection
relapse. The relationship between histological grading and tumor outcome
is unclear. Indeed, consistent histological grading of ependymomas has
proven difcult because a spectrum of pathological features exists, and the
distinction between classic and anaplastic is difcult. A clear international
consensus on this difcult issue is now required. Postoperative radiation
therapy to the tumor bed is an important component in the treatment of
localized ependymoma, and its role in the treatment of very young chil-
dren is changing. Although there is increasing evidence that a proportion
of ependymomas are chemoresponsive, a convincing role for chemotherapy
is still to be demonstrated. We still need to dene which drugs are active in
ependymoma. This in part reects the difculties in interpretation of post
chemotherapy imaging, and more sophisticated imaging studies are now
needed. Dening which patients are likely to benet from chemotherapy
is clearly an important albeit signicant challenge. Current recommenda-
tions for chemotherapy in ependymoma include (i) infants when the aim is
to delay or avoid radiotherapy and (ii) patients with residual tumor after
initial surgery. The systematic genetic analysis of tumor specimens obtained
following chemotherapy may provide useful information for future develop-
ment. A majority of tumor recurrences occur as a result of failure of local
tumor control and are identied between 9 and 24 months after therapy.
The prognosis for relapse is relatively poor, and overall only 25% of chil-
dren survive rst relapse. Better strategies for the management of relapse
are needed A number of important issues remain to be addressed. We need
to arrive at an international consensus on tumor grade, identify biological
correlates of outcome, devise imaging methods for assessing chemosensitiv-
ity, investigate which agents are active, and dene the role of newer radio-
therapy treatments. Signicant challenges for the years ahead.
254. ADVANCES IN MANAGEMENT OF
MEDULLOBLASTOMA
M. Grotzer; Division of Oncology, University Childrens Hospital of
Zurich, Zurich, Switzerland
Medulloblastoma is the most common malignant brain tumor of child-
hood, and yet it makes up only 1% of adult brain tumors. Medulloblastoma
is sensitive to chemotherapy and radiation, but successful surgical resection
continues to be an important component of therapeutic success. Advances
in the management of medulloblastoma have occurred in multiple areas
from improved neurosurgical techniques, rened dosing and delivery of
radiation, and optimized chemotherapy. Medulloblastomas are currently
risk-stratied as average risk or high risk depending on clinical factors such
as age, extent of resection, and presence of metastases. Molecular biol-
ogy and histological subtyping are beginning to improve prognostication
and may soon provide the means to accurately predict response to therapy.
Treatment for average-risk medulloblastoma has achieved a level of success
that allows efforts to be focused on the limitation of long-term sequelae
of therapy. Therapy for high-risk and relapsed medulloblastoma has been
positively affected by the advent of high-dose chemotherapy with peripheral
stem cell rescue. In addition, molecular targets are being elucidated and new
therapeutic agents are being tested for safety and efcacy.
255. ADVANCES IN THE MANAGEMENT OF CHILDHOOD
GLIOMAS: MOLECULAR MARKERS OF PROGNOSIS AND
NOVEL TREATMENT APPROACHES
I. Pollack; University of Pittsburgh, Pittsburgh, Pennsylvania, USA
With the exception of certain rare childhood glioma variants, pediatric
gliomas are histologically similar to lesions that arise in adults. However,
they have distinct molecular features, suggesting age-related pathways of
tumorigenesis. Whereas high-grade gliomas in adults, particularly grade IV
lesions, typically have amplication of EGFR and mutations of PTEN, such
changes are rare in pediatric high-grade gliomas. In contrast, approximately
40% of pediatric malignant gliomas have p53 mutations, which appear to
constitute an adverse prognostic factor. P16 or Rb deletions are observed
in approximately half of tumors, and correlate with p53 mutations. Unlike
the situation in adults, chromosome 1p and 19q deletions, although pres-
ent in a subset of gliomas, are not associated with a favorable prognosis.
Studies are in progress to determine whether prospective categorization of
tumors by these molecular features and markers of drug resistance identies
prognostically distinct tumor subgroups. In parallel with these analyses,
studies are in progress to test new therapeutic approaches for these tumors.
One approach that is being examined in several studies involves the use of
concurrent chemotherapy with irradiation, in addition to standard post-
radiation chemotherapy. Studies are also in progress that aim to counteract
drug resistance as a way of improving response to conventional chemothera-
peutic agents. A second general strategy involves targeting of the growth
signaling mediators that may contribute to tumor growth, such as PDGFR,
EGFR, and Ras, using small molecule inhibitors. A third approach incorpo-
rates targeted inhibition of pathways that contribute to tumor angiogenesis
and/or invasion. A fourth strategy applies convection-enhanced delivery of
high-molecular-weight macromolecules targeted to receptors that are over-
expressed on tumor cells compared to normal brain. Finally, it is likely that
increased understanding of the molecular features of childhood gliomas
will suggest additional relevant therapeutic targets.
256. RESPONSE OF ASYMPTOMATIC BRAIN METASTASES
OF SMALL CELL LUNG CANCER TO SYSTEMIC (FIRST-
LINE) CHEMOTHERAPY
T. Seute,
2
P. Leffers,
3
G.P.M. Ten Velde,
1
J.T. Wilmink,
4
and A.
Twijnstra
2
; Departments of
1
Pulmonology,
2
Neurology,
3
Epidemiology,
and
4
Radiology, University Hospital Maastricht, Maastricht, The
Netherlands
The objective of our study was to investigate the radiological response
of asymptomatic brain metastases (BM) from small cell lung cancer (SCLC)
to rst-line chemotherapy. BMs are a frequent and devastating complication
in SCLC patients. The standard treatment of symptomatic BM is whole-
brain radiotherapy (WBRT) in combination with corticosteroid medica-
tion. However, earlier studies found that BMs respond well to systemic
chemotherapy. It is postulated that the response rate (RR) of BM to systemic
chemotherapy reects the response of the primary tumor. Therefore, the
current prevailing opinion is that BM from SCLC should be treated initially
with systemic chemotherapy. Whereas previous reports studied the RR of
symptomatic BM to chemotherapy, the present study investigated the RR
of asymptomatic BM to rst-line chemotherapy. From 1980 to 2003, 462
consecutive patients with SCLC were enrolled in this study. Patients were
examined by a neurologist on a regular basis. Routine imaging of the brain
(CT or MRI) was performed before and after systemic chemotherapy. All
patients were initially treated with combination chemotherapy consisting
of cyclophosphamide, doxorubicin, and etoposide. Clinically manifest
BMs were treated with WBRT. Patients with asymptomatic BM did not
receive cranial irradiation. The RR of asymptomatic BM to chemotherapy
was assessed by changes in the size or the number of enhanced lesions on
MRI, using standard criteria. The MRI scans were blindly reviewed by an
experienced neuro-radiologist. We found 70 patients (15%) with BM at
diagnosis of SCLC. In 24 (5%) of these patients, BMs were asymptomatic.
The asymptomatic BMs were all diagnosed by MRI. Twenty-two patients
with asymptomatic BM completed ve cycles of chemotherapy and could be
evaluated for response. In six patients (27%) the BM responded (two com-
plete remissions, four partial remissions) to chemotherapy. In 16 patients
(73%) there was a systemic response. In ten patients the systemic response
was consonant with the cranial response. In 19 patients, BM became symp-
tomatic during follow-up, after completing chemotherapy, with a median
duration of 2.3 months. The progression-free survival did not differ among
cranial responders and non-responders. The median survival for patients
with asymptomatic BM was 8.3 months, whereas that for patients with
symptomatic BM was 8.0 months. The prevalence of asymptomatic BM in
SCLC is 5%. The RR of asymptomatic BM of SCLC to rst-line systemic
chemotherapy is 27%. We could not conrm the high RR found by for-
mer studies. Furthermore, the response of the BM was considerably worse
than the systemic response to chemotherapy. We therefore propose that the
treatment of BM from SCLC should consist of chemotherapy and cranial
irradiation.
257. BRAIN METASTASES FROM DIFFERENT TUMOR
TYPES: A SURVEY ANALYSIS FROM A MULTIDISCIPLINARY
EXPERIENCE
A. Fabi, A. Felici, A. Mirri, I. Sperduti, E. Bria, G. Lanzetta, G.
Mansueto, L. Moscetti, A. Pace, S. Telera, A. Vidiri, and C. Carapella;
Latium Neuro-Oncology Group, AINO, Regina Elena National Cancer
Institute, Rome, Italy
In order to evaluate trend and clinical behaviour of patients (pts)
affected by brain metastases (BM), the Latium Neuro-Oncology Group
members (Neurology, Neurosurgery, Oncology, and Radiotherapy Units)
applied a multi-institutional survey to clarify the commonly employed
therapeutic strategies and to indicate the most effective approach arising
from a multidisciplinary experience. Primary outcomes have been iden-
tied the median overall survival (OS) and median overall survival after
BM appearance (BM-OS). The outcome monitoring was accomplished
through a questionnaire with several items regarding patients character-
istics and therapeutic approach to BM from different primary tumors. From
March 2003 to November 2004, 203 patients were registered for the study.
The median age was 60 (range, 2088), male/female distribution 107/96
(53%/47%). The primary tumor sites were as follows: NSCLC 98 (48%),
breast 44 (22%), colorectal 23 (11%), melanoma 18 (9%), SCLC 8 (4%),
unknown primary tumor 7 (3.5%), kidney 2 (1%), others 3 (1.5%). Sites
of BM were as follows: supratentorial in 118 pts (58%), infratentorial in 54
(27%), and infra-supratentorial in 31 (15%). Neurological symptoms were
present in 118 pts (58%), and the RPA-RTOG scale was I for 70 pts (34%),
II for 115 pts (57%), III for 12 pts (6%), and unknown for 6 pts (3%). One
lesion was observed in 90 pts (44%), 2 to 3 lesions in 35 pts (17%), and
more than 3 lesions in 78 pts (39%). First- and second-line treatment for
BM was given at 197 and 87 pts, respectively. Surgery was the rst- and
second-line treatment in 46 (23%) and 8 (9%) pts, chemotherapy in 45
(23%) and 40 (46%) pts, whole brain radiation (WBRT) in 91 (46%) and
35 (40%) pts, and radiosurgery in 14 (7%) and 4 (5%). At a median fol-
low-up of 18.5 months, the median OS was 11 (815) months; with respect
to different tumor type the 1-year survival rate was lung 52.5%, breast
47.7%, melanoma 30%, and colorectal 12.7%. Pts with 1 and 2 lesions
present a median OS of 16 (924) months and 7 (59) months, respectively
(P 0.0005). Local aggressive treatment (surgery and radiosurgery),
mainly considered in pts with 1 to 3 lesions and limited systemic disease,
was demonstrated to be superior in terms of median OS compared with CT
and WBRT (17 and 9 months, P 0.004). In our survey, no signicant
differences were seen in OS from BM diagnosis between patients affected
by either lung or breast cancer, but a signicant OS difference was observed
for colorectal cancer pts in which BM presents the worst prognosis. Num-
bers of lesions, limited systemic disease, and local aggressive treatment are
predictive factors for OS.
258. LEPTOMENINGEAL METASTASIS: A DESCRIPTIVE
META-ANALYSIS OF 2765 PATIENTS
L. Junck; Neurology, University of Michigan Hospital, Ann Arbor,
Michigan, USA
The purpose of this study was to characterize leptomeningeal metastasis
by meta-analysis of published series. The literature was searched for articles
describing series of 18 or more patients with multiple primary malignancies,
mainly or entirely adults, published between 1974 and 2004 in English.
Each analyzed feature, e.g., proportion of cases with each type of primary
malignancy, was normalized to the appropriate subset of patients. Results
were compared to U.S. statistics for all cancer deaths from the SEER registry.
Forty-nine series were found. Among common malignancies, the primary
malignancy was as follows: breast 24%, leukemia 20%, lymphoma 17%,
lung 14%, CNS 11%, melanoma 5%, colorectal 0.7%, prostate 0.4%. An
index of relative incidence of leptomeningeal metastasis was calculated in
relation to all cancer deaths, adjusted so that the incidence averages 1.0
for all cancers. This relative incidence is as follows: leukemia 5.1, CNS
4.9, lymphoma 3.9, melanoma 3.6, breast 3.1, lung 0.51, prostate 0.08,
colorectal 0.06. Among patients with lung cancer, adenocarcinoma and
small-cell carcinoma are overrepresented, accounting for 45% and 35% of
cases of leptomeningeal metastasis, respectively, though these subtypes each
account for a small minority of lung cancer cases. Average survival, includ-
ing both treated and untreated cases, was 2.6 months. Among patients who
underwent lumbar puncture (LP), the initial positive cytology was obtained
with the rst LP in 73%, the second LP in 22%, and the third LP in only
3%. CSF analysis revealed increased WBC in 68%, decreased glucose in
47%, increased protein in 82%, and normal analysis in 6%. The risk of lep-
tomeningeal metastasis varies widely by cancer type. Although lung cancer
is considered a common source, the risk of leptomeningeal metastasis with
lung cancer in relation to cancer death is less than 1/6 that of several other
types of malignancy. Survival is poor. Performing CSF cytology on two
LPs adds to diagnostic yield, but a third LP rarely helps. While CSF WBC,
glucose, and protein are non-specic, some of the ndings are nearly always
abnormal and can aid in diagnosis.
259. A PHASE 2 TRIAL OF INTRA- CSF ETOPOSIDE IN THE
TREATMENT OF NEOPLASTIC MENINGITIS
M. Chamberlain; Interdisciplinary Oncology, H. Lee Moftt Cancer
Centre, Tampa, Florida, USA
The objective of this study was to determine the toxicity and response
rate of intra-CSF etoposide [VP-16] in the treatment of patients with neo-
plastic meningitis [NM]. NM, a metastatic complication of both primary
CNS and systemic cancer, occurs in 1% to 5% of patients with known
cancer. Currently available treatment options are limited and provide only
modest benefit. Twenty-two patients (median age 55 years) with clini-
cal and cytologically documented NM received intra-CSF VP-16. Tumor
histologies included the following: lung (6 patients), breast (4), brain (4),
non-Hodgkins lymphoma (3), melanoma (3), colon (1), and prostate (1).
Concurrent involved-eld radiotherapy (16/22 patients) or systemic chemo-
therapy (15/22) was administered based on clinical indications. VP-16 was
administered at a xed dose (0.5 mg every day given 5 times per week every
other week for 8 weeks [induction]). Patients were evaluated by CSF cytol-
ogy and neurological examination at the conclusion of induction therapy.
Responding patients continued to receive VP-16 (5 consecutive days every
4 weeks) with monthly evaluations. Seven of 22 patients (32%) treated with
VP-16 had a cytological response and either stable or improved neurological
status at the conclusion of induction. Six of 22 patients (27%) progressed
during induction therapy and did not receive the 8-week induction course
of therapy. In responding patients, duration of response ranged from 8 to
40 weeks (median 24 weeks). Toxicity was manifested as transient chemi-
cal arachnoiditis (4/22 patients; 10% of all treatment cycles). There was no
evidence of myelosuppression nor were treatment-related hospitalizations
or deaths seen. It is concluded that VP-16 has modest activity against NM
and easily managed toxicity.
260. PHASE 3 STUDY COMPARING RADIOTHERAPY
WITH SUPPORTIVE CARE IN OLDER PATIENTS WITH
NEWLY DIAGNOSED ANAPLASTIC ASTROCYTOMAS (AA)
OR GLIOBLASTOMA MULTIFORME (GBM): AN ANOCEF
GROUP TRIAL
F. Keime-Guibert, O. Chinot, L. Taillandier, S. Cartalat-Carel,
M. Frenay, G. Kantor, J. Guillamo, E. Jadaud, P. Colin, P. Bondiau,
P. Mene, H. Loiseau, V. Bernier, J. Mazeron, J. Honnorat, M. Barri,
A. Bissery, and J. Delattre; Association des Neuro-Oncologues
dExpression Franaise
Despite evidence of increased incidence, the optimal treatment of malig-
nant astrocytomas in elderly patients is unsettled, ranging from palliative
care to a vigorous approach with radiotherapy and chemotherapy. To start
evaluating this issue, patients with AA or GBM, age 70 years or older, and
a Karnofsky performance status of at least 70 were randomly assigned after
biopsy or surgical excision to receive either supportive care (corticosteroids,
anticonvulsants, physical therapy, and palliative support) or supportive care
and focal RT (50 Gy/28 fractions/38 days). Randomization was stratied
by center. The primary end point was overall survival. The secondary end
points were tolerance and quality of life. Between February 2001 and
December 2004, 84 patients with a median age of 73 years (range, 7085)
were randomized in 11 medical centers. A sequential triangular design was
used. and the trial reached a stopping boundary at the rst interim analysis
(P 0.004). Median survival of the 40 patients who received RT was 28
weeks compared with 17 weeks for the 44 patients who had only supportive
care. There were no clinically severe adverse reactions related to RT. Qual-
ity of life evaluation is ongoing. This trial, which is the rst phase 3 study
specically devoted to the management of malignant astrocytomas in the
elderly (age 70 years or more), demonstrates that focal RT increases survival
in this population.
261. THE ROLE OF FRACTIONATED STEREOTACTIC
RE-IRRADIATION IN RECURRENT GLIOMAS
S. Combs, S. Gutwein, D. Schulz-Ertner, C. Thilmann, P. Huber, and
J. Debus; Radiation Oncology, University of Heidelberg, Heidelberg,
Germany
The present analysis evaluates the effectiveness of fractionated stereo-
tactic re-irradiation (FSRT) in recurrent gliomas. From January 1995 to
July 2003, 156 patients with recurrent gliomas were treated with FSRT. At
primary diagnosis, 63 patients had WHO grade II tumors, WHO grade III
astrocytomas were diagnosed in 40 patients, and 53 patients suffered from
glioblastoma multiforme (GBM). All patients underwent neurosurgical
interventions at primary diagnosis. Median time between primary diag-
nosis and re-irradiation was 50, 31.5, and 10 months for grade II, III, and
GBM, respectively. Using 3-4 non-coplanar elds formed with a multi-leaf-
collimator, a median dose of 36 Gy was applied in a median fractionation
of 5x2 Gy/week depending on the size and the localization of the lesion.
No concomitant chemotherapy was applied. Radiation was well tolerated
by all patients. No severe side effects occurred. Median overall survival was
111 months for patients with grade II gliomas, 48 months for grade III, and
21 months for patients with GBM. Calculated from the initiation of FSRT,
median survival was 23, 16, and 8 months for grade II, III, and IV gliomas.
Main prognosticators for overall survival were histology, and extent of neu-
rosurgical resection. We conclude that stereotactically guided fractionated
re-irradiation is a safe and effective treatment modality in selected cases
with recurrent gliomas. Further investigations to continuously improve
overall survival in patients with astrocytomas are warranted, especially
with regard to newer radio-chemotherapeutic strategies.
262. LONG-TERM FOLLOW-UP OF PATIENTS WITH
PRIMARY CENTRAL NERVOUS SYSTEM LYMPHOMA
TREATED WITH A HIGH-DOSE METHOTREXATE-
BASED REGIMEN WITH OR WITHOUT WHOLE BRAIN
RADIOTHERAPY
I. Gavrilovic, A. Hormigo, A. Kim, J. Yahalom, L. DeAngelis, and L.
Abrey; Neurology, Memorial Sloan-Kettering Cancer Center, New York,
New York, USA
The primary objective of this study is to report the long-term outcome
of newly diagnosed primary central nervous system lymphoma (PCNSL)
patients treated with high-dose methotrexate (MTX)-based chemotherapy
with or without whole brain radiotherapy (WBRT). Our initial report of
this regimen (J. Clin. Oncol. 18, 3144, 2000) projected an overall median
survival of 60 months and an excellent outcome for patients younger than
age 60. We have now followed all surviving patients for nearly 10 years and
have detailed information regarding relapse and delayed treatment toxicity.
There were 57 patients with a median age of 65 years (range, 2289 years)
and median Karnofsky performance status of 70 (range, 30100). Patients
received ve cycles of MTX 3.5 g/m
2
and vincristine 1.4 mg/m
2
. Procarba-
zine 100 mg/m
2
/d for 7 days was given with the rst, third, and fth cycle
of MTX. Three weeks after WBRT or cycle 5 of MTX, cytosine arabinoside
3 g/m
2
was given for two cycles for a total of 4 doses. Thirty-one patients
received WBRT (45 Gy) and 26 older patients deferred WBRT in an effort
to reduce treatment-related neurotoxicity. Thirty-seven patients have died,
and the overall median survival was 51 months with a median follow-up of
the 20 surviving patients 115 months (range, 12144 months). Recurrent
disease developed in 22/57 patients (39%) typically within 3 years of diag-
nosis; one patient developed an isolated systemic non-Hodgkin lymphoma
7.4 years after PCNSL diagnosis. Thirty percent (17/57) of all patients have
developed treatment-related dementia; the risk was higher in patients age 60
and older who received WBRT (75%), but 26% of those patients 60 who
received WBRT also developed cognitive decits or dementia. Late neuro-
toxicity has not developed in any surviving patient who deferred WBRT.
At last follow-up, a total of 20/57 patients (35%) are alive, including 74%
of the patients under the age of 60 at diagnosis plus 19% of older patients
who received chemotherapy alone. Cause of death was tumor in 23 patients
(40%), acute toxicity during salvage treatment in 2 (3.5%), late toxicity in 9
(16%), other cancers in 2 (3.5%), and unknown in one (1.8%). Long-term
follow-up conrms our initial observation of excellent overall survival with
this regimen. Younger patients do particularly well, but have a signicant
risk of developing late neurotoxicity. A subset of older patients who deferred
WBRT enjoyed prolonged survival and an excellent quality of life without
evidence of treatment-induced neurotoxicity.
263. MODIFIED BORON NEUTRON CAPTURE THERAPY
(BNCT) FOR MALIGNANT GLIOMAS USING EPITHERMAL
NEUTRON AND TWO BORON COMPOUNDS WITH
DIFFERENT ACCUMULATION MECHANISMS:
EFFECTIVENESS OF BNCT ON RADIOGRAPHIC IMAGES
S. Miyatake
1
, Y. Kajimoto
1
, S. Kawabata
1
, K. Yokoyama
1
, K. Kuroiwa
1
,
and O. Ono
2
;
1
Neurosurgery, Osaka Medical College, Takatsuki;
2
Radiation Oncology, Kyoto University, Research Reactor Institute,
Kumatori; Japan
Boron neutron capture therapy (BNCT) is tumor-specic radiotherapy.
To improve the effectiveness of BNCT for malignant gliomas, we utilized
epithermal neutron and two different boron compounds, sodium borocap-
tate (BSH) and boronophenylalanine (BPA) with different accumulation
mechanisms to increase the boron level in tumors. Thirteen patientsten
glioblastoma, one gliosarcoma, one anaplastic astrocytoma, and one ana-
plastic oligoastrocytoma (6 were new and 7 were recurrent)were treated
with this modied BNCT from January 2002 to December 2003. Only one
glioblastoma patient had no postoperative enhanced lesion. The patients
received
18
F-labeled BPA PET to assess the accumulation and distribution
of BPA before neutron irradiation. Irradiation time was determined not to
Abstracts from the Eleventh International Symposium on Pediatric Neuro-Oncology
350 Neuro-Oncology OCTOBER 2004
exceed 13 Gy-Eq to the normal brain and as much as we could to contrast
enhanced tumor for the new cases. For the recurrent cases, irradiation time
was determined in each case according to the previous irradiation dose and
eld. Five grams of BSH and 250 mg/kg of BPA were administrated 12 h
and 1 h before neutron irradiation, respectively. The patients received volu-
metric assessments, by MRI or CT scanning. Improvements in the images
were assessed at 2 to 7 days after irradiation as initial effects, and their
maximum effects on serial radiographic images were also analyzed. Sur-
vival period was also estimated after diagnosis and BNCT. Six cases out
of 13 were analyzed the peak activity of chorine and N-acetyl aspartate
(NAA) by MR spectroscopy (MRS) to analyze tumor-specic cytotoxic-
ity and neuronal preservation The lesion/normal brain (L/N) ratio of BPA
before BNCT on PET varied from 2.65 to 7.8. There was a tendency of
low L/N ratio in recurrent cases and high in primary cases. The neutron
irradiation time varied from 60 to 120 min. The mean of initial tumor
volumes prior to BNCT was 42.3 (2.2107.6) cm
3
. Irrespective of initial
tumor volume, in all patients who had assessable lesions, the improvements
on MRI/CT images were recognized both on initial assessments (volume
reduction rate: 17.4%71.0%, mean 46.4%) and on follow-up assessments
(30.3%87.6%, mean 58.5%). More than 50% of the contrast-enhanced
lesions disappeared in 8 out of the 12 patients during the follow-up period.
Mean survival after the diagnosis was over 19 months. Three patients are
still alive. Four cases were lost by CSF dissemination, and 2 cases were lost
by other cause of death. MRS showed preserved NAA activity while Cho-
rine activity decreased after BNCT, which showed tumor-specic destruc-
tion and neuronal preservation by this treatment. By this BNCT method,
good improvements of malignant glioma patients on radiographic images
and improved survival were obtained, and tumor-specic destruction was
proven.
264. PRELIMINARY RESULTS OF A PHASE 2 STUDY OF
TP-38 IMMUNOTOXIN DELIVERED VIA CONVECTION-
ENHANCED DELIVERY TO PATIENTS WITH RECURRENT
GLIOBLASTOMA MULTIFORME: REPORT OF THE TP-38
STUDY GROUP
F. Weber; Neurosurgery, Klinikum Saarbrcken, Saarbrcken, Germany
TP-38 is a recombinant chimeric protein composed of the epidermal
growth factor receptor (EGFR) binding ligand (TGF-a) and a genetically
engineered form of the Pseudomonas exotoxin PE-38. We report prelimi-
nary results of a randomized, phase 2 study conducted at multiple European
centers. Either of two dose levels of TP-38 (50 ng/ml or 100 ng/ml) was
administered to patients with recurrent glioblastoma in a single treatment
consisting of a continuous, intratumoral infusion. This was a nonresec-
tion study; patients did not undergo tumor resection immediately prior
to treatment. Three catheters were stereotactically placed in investigator-
determined locations within the enhancing tumor area. The infusion rate
was 0.2 ml/min per catheter. Each catheter delivered 13.4 ml over 67 h.
The total volume infused was approximately 40 ml, and the total dose of
TP-38 infused was approximately 2 g or 4 g. Patients were followed until
death. Tumor responses were assessed by MRI at every 8 weeks, beginning
4 weeks after treatment. Safety was closely evaluated. Time to progression,
progression-free survival, and overall survival were measured end points.
Thirty-four of the planned 38 patients have been treated thus far. Safety and
tolerability has been excellent. The 4-week and 12-week post-infusion MRI
scans often showed treatment-related changes that make response assess-
ment difcult. These changes usually resolved by 20 weeks post treatment
MRI. Preliminary data show that one patient has a complete response 48
weeks after infusion, and another patient showed a partial response over
60 weeks, 24 patients remained stable. These results in a not highly selected
group of patients suffering from recurrent glioblastoma are promising, and
we are proceeding with a second trial where the tumor gets rst resected
and the tumor adjacent area will be treated by CED of TP38.
265. IMMUNOLOGIC TARGETING OF THE TUMOR-
SPECIFIC ANTIGEN CONTAINED IN THE DELETION
MUTATION OF THE EPIDERMAL GROWTH FACTOR
RECEPTOR (EGFRVIII) WITH PEPTIDE LOADED
DENDRITIC CELLS IN PATIENTS WITH MALIGNANT
GLIOMAS
G. Archer,
1
D. Bigner,
1
A. Friedman,
1
H. Friedman,
1
K. Penne,
1
A.
Paolino,
2
D. Smith,
1
D. Reardon,
1
and J. Sampson
1
;
1
Duke University
2
Emory University, Atlanta,
Georgia; USA
The use of immunotherapy for the treatment of malignant gliomas
requires the generation of a strong immune response in what has tradition-
ally been considered an immunologically privileged site. Most importantly,
the immune response must be specic for the tumor so as not to cross-react
with normal brain, which could result in a devastating autoimmune enceph-
alomyelitis. The deletion mutation of the epidermal growth factor receptor
(EGFRvIII) is a tumor specic antigen, which is expressed on approxi-
mately 47% of all malignant gliomas (MGs). The in-frame EGFRvIII dele-
tion combines distant parts of the molecule producing a novel glycine at the
fusion junction. We have initiated a phase 1 clinical trial for patients with
newly diagnosed MGs to determine the safety of vaccinating with mature
DCs loaded with a peptide spanning the fusion junction of the EGFRvIII
conjugated to keyhole limpet hemocyanin (PEPvIII-KLH). The vaccina-
tion protocol consist of 3 vaccines 2 weeks apart of PEPvIII-KLH loaded,
mature DCs beginning 2 weeks following completion of post-resection
radiotherapy. To date, 19 patients have been enrolled with 16 completing
vaccination with no adverse events. No patient showed a positive delayed-
type hypersensitivity reaction to KLH or PEPvIII before vaccination and
of the patients who could be evaluated after vaccination, 14/15 patients
(93.3%) reacted to KLH and 11/15 (73.3%) reacted to PEPvIII. In vitro
proliferation in response to PEPvIII was seen in 11/12 (92%) and to KLH
in 9/12 (75%) of patients tested. At two weeks following the completion
of the vaccination protocol, no humoral response to PEPvIII was found.
Two patients, one with anaplastic astrocytoma and one with glioblastoma
multiforme (GBM) with residual radiographic disease after resection, and
radiation, have had a nearly complete response following completion of
vaccination. These patients have remained stable for 921 and 1216 days.
Of the 14 patients without radiographically evident disease, 4/14 (28.6%)
have not progressed at 418, 914, 964, 2738 days with a median overall time
to progression of 315 days. For patients with GBM, the median survival
time was 609 days, which compares favorably with recently published trials
evaluating newly diagnosed patients with GBM treated with Gliadel (417
days [Westphal et al., Neuro-Oncology, 79, 2003]), radiation and concur-
rent Temodar (409 days [Stupp et al., ASCO June 2004]), or radiolabeled
anti-tenascin monoclonal antibodies (556 days [Reardon et al., J. Clin.
Oncol. 1389, 2002]). These ndings suggest that autologous mature DCs
loaded with the tumor-specic antigen PEPvIII are safe and may induce a
benecial immunologic response in patients with MGs.
266. PHASE 1/2 CLINICAL TRIAL OF CTL PRECURSOR-
ORIENTED PERSONALIZED PEPTIDE VACCINATION
THERAPY FOR PATIENTS WITH RECURRENT GLIOMA
R. Yamanaka
1
, N. Yajima
1
, N. Tsuchiya
1
, J. Honma
1
, M. Sano
1
,
Y. Obata
3
, M. Shigemori
2
, T. Mine
3
, K. Itoh
3
, and R. Tanaka
1
;
1
Neurosurgery, Brain Research Institute, Niigata University, Niigata;
2
Neurosurgery and
3
Immunology, Kurume University School of
Medicine, Kurume; Japan
The purpose of this study was to investigate the safety and immunologi-
cal responses of personalized peptide vaccination in patients with malignant
glioma. Twenty-six patients with recurrent malignant glioma entered in the
phase 1 clinical study of personalized peptide vaccination. Peripheral blood
mononuclear cells (PBMCs) and plasma prior to vaccination were provided
for cellular and humoral responses in vitro to each of 31 or 36 peptides in
cases of HLA-A24 or HLA-A2 patients, respectively, and then only the
reactive peptides (maximum: 4) were allowed to in vivo administration.
Post-vaccination PBMCs and plasma, and also pre-and post-vaccination
cerebrospinal uid, were provided for their reactivity to the vaccinated pep-
tides. The protocol was generally well tolerated, although the majority of
patients developed grade 1 or 2 local redness and swelling at the injection
site. Increase in cellular and humoral responses specic to at least one of
the vaccinated peptides was observed in the post-vaccination (6th) PBMCs
and plasma from 62% and 73%, respectively. More importantly, peptide-
specic IgG were found in the post-vaccination cerebrospinal uid of tumor
sites. Clinical responses were 4 cases with partial response, 9 with stable
disease, and 10 with progressive disease. Mean survival time has not been
reached at the mean observation time 217 days. Personalized peptide vac-
cination is well tolerated and has the ability to induce immune responses
in the majority of malignant glioma patients, along with several cases of
major tumor regression. These results would encourage the phase 2 clini-
cal study of personalized peptide vaccination for patients with recurrent
malignant glioma.
267. THE TGF-BETA2 SPECIFIC ANTISENSE
OLIGONUCLEOTIDE AP 12009 AS IMMUNOTHERAPY IN
HIGH- GRADE GLIOMA: A CLINICAL PHASE IIB STUDY
P. Hau,
1
W. Grisold,
2
M. Mahapatra,
3
M. Mehdorn,
4
V.E. Parfenov,
5

J. Pichler,
6
M. Zaaroor,
7
U. Bogdahn,
1
K.-H. Schlingensiepen,
8
and the
AP 12009 glioma study group;
1
Clinic and Policlinic for Neurology,
University Regensburg, Regensburg, Germany;
2
Kaiser Franz Josef
Spital, Neurologische Ambulanz, Wien, Austria;
3
All India Institute of
Medical Sciences, Neurosciences Centre, New Delhi, India;
4
Klinik fr
Neurochirurgie, Universittsklinikum Kiel, Kiel, Germany;
5
Department
of Neurosurgery, Military Medical Academy, St. Petersburg, Russia;
6
Department of Neurosurgery, Landesnervenklinik Wagner-Jauregg,
Linz, Austria;
7
Department of Neurosurgery, Rambam Medical Center,
Haifa, Israel;
8
Antisense Pharma GmbH, Regensburg, Germany
Transforming growth factor-beta2 (TGF-beta2) is an important factor
in malignant progression by inducing proliferation, invasion, metastasis,
and angiogenesis. Furthermore, TGF-beta is the most potent immuno-
suppressor known. High-grade (malignant) gliomas are highly aggressive
tumors characterized by distinct overexpression of TGF-beta2, responsible
for the immunodecient state of glioma patients, especially in advanced
stage. AP 12009, a phosphorothioate antisense oligodeoxynucleotide spe-
cic for the human TGF-beta2 mRNA, has been developed as a targeted
anti-tumor therapy and already has proven safety and shown anti-tumor
activity in phase 1/2 clinical studies in recurrent high-grade glioma. Based
on these results, an open-label active-controlled phase 2b multi-national
study in adult patients with recurrent high-grade glioma has been initi-
ated and is currently ongoing. Patients are randomized into 3 treatment
groups to receive one of two doses of AP 12009 or standard chemotherapy,
i.e., temozolomide (TMZ), or if patients had already received TMZ before
the combination regimen procarbazine/CCNU/vincristine (PCV). Primary
objectives are the response rate (RR), progression-free survival (PFS), and
overall survival at different time points. AP 12009 is administered intra-
tumorally as continuous high-ow microperfusion over a 7-day period
every other week for up to six months. Both efcacy and safety will be used
as criteria for evaluation. In the phase 1/2 studies with 24 patients, the
median overall survival time was longer as compared to recent literature
(Yung et al., 2000; Theodosopoulus et al., 2001; Chang et al., 2004) on
standard chemotherapy. Response data after AP 12009 in phase 1/2 include
several patients with stabilizations and two patients with long-lasting com-
plete tumor remissions. As of November 2004 more than 120 patients have
been enrolled in the current phase 2b study.
268. PRE-RADIATION CHEMOTHERAPY MAY IMPROVE
SURVIVAL RATE OF DIFFUSE BRAINSTEM GLIOMAS:
FINAL RESULTS OF BSG98 TRIAL
D. Frappaz,
1
M. Schell,
1
P. Marec-Berard,
1
C. Mottolose,
2
D. Perol,
1

C. Bergeron,
1
T. Philip,
1
A. Ricci,
2
S. Galand-Desme,
1
A. Szathmari,
2

and C. Carrie
1
;
1
Neuro-oncology, Centre Lon Brard, Lyon;
2
Hpital
Wertheimer, Lyon; France
Despite numerous attempts (standard RT, hyperfractionated RT, stan-
dard or high-dose chemotherapy), the usual median survival of patients
with diffuse brain stem tumor does not exceed 9 months. We prospectively
proposed to delay radiotherapy as long as no progression was observed
under multidrug chemotherapy. As soon as MRI showed a diffuse BSG,
cycles of chemotherapy were initiated. Each cycle included three monthly
courses, alternating BCNU and CDDP (40 mg/m/d 4 days in continuous
infusion), course and high-dose MTX (12 g/m). Cycles were delivered until
progression, or to a maximum of 4 (1 year of treatment). Standard radiation
therapy was then delivered to the tumor with maintenance hydroxyurea.
Twenty-three patients were prospectively included. They were compared
with a historical control group of 14 patients treated in the same institu-
tion, by front-line radiotherapy only and/or procarbazine or carboplatin.
The median number of cycles was 3 (1 to 4). Iatrogenic severe infections
occurred in 4 patients, and 11 patients required platelet transfusions. The
median survival is signicantly increased as compared to historical con-
trols (17 months [95% CI, 1023 months] vs. 9 months [95% CI, 810
months]; P 0.015), and survival from time of radiotherapy is similar
in both groups, though the number of days in hospital was prolonged (57
vs. 25 days; P 0.001). Steroids could be at least transiently withdrawn
in half of the patients of both groups (P 0.79). We conclude that front-
line chemotherapy alternating hematotoxic and nonhematotoxic schedules
signicantly increases overall median survival. However, the price to be
paid (infections and hospitalization) deserves honest discussion with the
children and their parents.
269. OUTCOME OF INTENSIVE INDUCTION
CHEMOTHERAPY FOLLOWED BY CONSOLIDATIVE
MYELOABLATIVE CHEMOTHERAPY AND AUTOLOGOUS
STEM CELL RESCUE (AUSCR) IN YOUNG CHILDREN WITH
NEWLY DIAGNOSED NON- CEREBELLAR PRIMITIVE
NEUROECTODERMAL TUMORS (PNET): THE HEAD
START I AND II PROTOCOLS
J. Finlay,
1
J. Allen,
2
B. Diez,
3
S. Kellie,
4
S. Chi,
5
S. Asgharzadeh,
1
S.
Zacharoulis,
1
and S. Gardner
2
;
1
Neural Tumors Program, Childrens
Hospital Los Angeles, Los Angeles, California, USA;
2
Pediatric
Oncology, New York University, New York, New York, USA;
3
Neuro-oncology, Institute FLENI, Buenos Aires, Argentina;
4
Pediatric
Oncology, The Childrens Hospital at Westmead, Westmead, Australia;
5
Pediatric Neuro-Oncology, Dana Farber Cancer Institute, Boston,
Massachusetts, USA
The outcome for young children with non-cerebellar PNET has been
dismal with conventional chemotherapy attempting to either delay or avoid
irradiation. Between 1991 and 2002, 42 children (median age 2.9 years;
range, 0.27 years) were treated with two serial studies: Head Start I
(19911997) and Head Start II (19972002) in an effort to avoid or
reduce irradiation. Head Start I induction chemotherapy included 5 cycles
of vincristine, cisplatin, cyclophosphamide, and etoposide at 3- to 4-week
intervals, followed by consolidation with thiotepa, carboplatin, and etopo-
side with AuSCR. Head Start II therapy was identical, except for the addi-
tion of high-dose (400 mg/kg) methotrexate during each induction cycle for
patients with metastatic (M1) disease. No irradiation was administered to
patients. The 1-, 2-, and 5-year Kaplan Meier analyses of event-free survival
(EFS) are 66.2 4.4, 42.8 7.9, and 40%, and of overall survival (OS)
are 71 7, 49.6 8.6, and 43.4%. A signicant difference in outcome was
noted for children 36 months; 1- and 5-year OS for children 36 months are
74% and 57%. A trend for improved survival with radical resection was
noted: 5 of 13 patients with less than a radical resection survive without
disease compared with 14 of 24 with radical resection. All 4 children with
brainstem PNET died of tumor progression. Patients with M1 disease at
diagnosis fared poorly: Of 5 pineoblastoma, 1 brainstem PNET, and 1 other
supratentorial PNET, two survive without disease. The outcome for chil-
dren with non-cerebellar PNET with this intensive chemotherapy strategy
still remains poor for children.
270. SALVAGE TREATMENT FOR EPENDYMOMA (EPD)
AFTER SURGERY ONLY: PITFALLS OF OMITTING AT
ONCE ADJUVANT TREATMENT
M. Massimino,
1
L. Gandola,
1
F. Giangaspero,
2
A. Sandri,
2
M. Garr,
2

L. Genitori,
2
G. Perilongo,
2
G. Scarzello,
2
F. Spreaco,
1
M. Mascarin,
2

P. Modena,
1
and E. Madon
2
;
1
Istituto Nazionale Tumori, Milano, Italy;
2
AIEOP Neuro-Oncology group
From 1993 to 2002, while 63 children were electively treated with the
Italian post-surgical protocol for EPD (hyperfractionated radiotherapy
[HFRT] VCR/CTX/VP16), 14 other patients were referred for adjuvant
treatment after 2 (n 12) or 3 (n 2) excisions of the tumor whose treat-
ment had been considered exclusively surgery by the referral neurosurgeon.
Mean time to local progression had been 14 months, mean age at diagnosis,
5 years. Tumor originated in posterior fossa (PF) in 10 children and was
supratentorial (ST) in the other 4; 11 tumors had been completely excised
at both surgical report and radiological evaluation, 3 had macroscopic resi-
dues. Histological diagnosis was classic EPD in 9 pts and anaplastic in 5.
Eight children were referred NED and 6 ED after second or further surgery,
5 had low cranial nerves palsy (1 requiring tracheostomy), 1 had recur-
rent surgery-related meningitis, and 2 had persistent hydrocephalus. All
children were treated with RT to tumor bed (6 HFRT: 70.4 Gy; 8 standard
RT: 5459.4 Gy) and 5 also with pre-RT CT. Five of 14 pts (5/10 with PF
tumors) had a further relapse at a mean of 6 months after last surgery; all
have died, thus obtaining a PFS of 66% and an OS of 74% at a mean fol-
low-up of 3 years after referral. Considering only pts with PF tumors, PFS
and OS were 53% and 61%, respectively. Patients with relapsing EPD after
surgery only, especially if originating in PF, have a more severe prognosis
despite surgery completeness and non-anaplastic subtype than pts receiving
adjuvant treatment after rst diagnosis (PF tumors 3 years, PFS: 53% vs.
63%, P 0.05); moreover, subsequent surgical acts for tumor re-growth
are followed by severe neurological sequelae.
271. OUTCOME OF INTENSIVE INDUCTION
CHEMOTHERAPY FOLLOWED BY CONSOLIDATIVE
MYELOABLATIVE CHEMOTHERAPY AND AUTOLOGOUS
STEM CELL RESCUE (AUSCR) IN YOUNG CHILDREN WITH
NEWLY DIAGNOSED EPENDYMOMA
S. Zacharoulis,
1
A. Levy,
4
S. Gardner,
2
I. Dunkel,
3
J. Allen,
2
S.
Asgharzadeh,
1
and J. Finlay
1
;
1
Pediatric Neuro-Oncology, Childrens
Hospital Los Angeles, Los Angeles, California;
2
Pediatric Oncology,
New York University, New York, New York;
3
Pediatric Oncology,
Memorial Sloan- Kettering Cancer Center, New York, New York;
4
Pediatric Oncology, Monteore Medical Center, New York, New York,
USA
The outcome of young children with ependymoma has been dismal with
conventional chemotherapy attempting to either delay or avoid irradiation.
This study investigates the efcacy of high-dose chemotherapy followed
by stem cell rescue in pediatric patients with ependymoma. The patients
were 28 children from Head Start I (19911997) and Head Start II
(19972002). Overall, the mean age at diagnosis was 2.4 years. Twenty-
four patients with local disease received an induction regimen of 5 cycles
of chemotherapy (cisplatin, vincristine, etoposide, cyclophosphamide at
34 week intervals). All four patients with leptomeningeal dissemination
received the same drugs with the addition of high-dose methotrexate and
leucovorin rescue. After induction, individuals in both groups without evi-
dence of disease proceeded to marrow ablative chemotherapy (thiotepa, car-
boplatin and etoposide) with autologous stem cell rescue. Twenty patients
(71%) had posterior fossa tumors and eight supratentorial tumors. Seven-
teen patients (61%) had a gross total resection (GTR) at diagnosis. The 1-,
2-, and 5-year Kaplan Meier analyses of progression-free survival (PFS) of
these patients are 70 9%, 43 10%, and 14 7%. The 1-, 2-, and 5-year
overall survival (OS) are 75 8%, 63 9%, and 38 10%. Survival data
did not differ signicantly for patients 3 years old at diagnosis. Thirty-seven
percent of the patients with supratentorial tumors are long-term survivors
compared to 55% of the patients with posterior fossa tumors. Half of the
patients who underwent a GTR survived compared to 36% of the ones
for whom GTR was not achieved. Radiation was administered to 15/28
patients overall: 75% of patients with prolonged survival received RT. Two
of the four patients with disseminated disease are long-term survivors. The
toxic mortality within this group of 28 patients was 14%. Outcome with
this treatment strategy does not appear superior to other strategies seeking
to avoid or delay RT.
272. GENE THERAPY
J. Yoshida; Department of Neurosurgery, Nagoya University Graduate
School of Medicine, Nagoya, Japan
Gene therapy is a new medicine based on a technique for correcting
defective genes responsible for disease development. It holds potential for
treating or even curing formidable diseases such as monogenic metabolic dis-
orders, cancers, infectious diseases, and vascular diseases. More than 1000
clinical trials involving 6000 patients were identied worldwide in 2004,
but almost all trials are aimed at establishing the safety of gene therapy
rather than the effectiveness. In fact, gene therapy still faces many scientic
or ethical obstacles before it can become a good practical medicine for man.
A major blow to disturb the success of gene therapy came in 1999, which is
the death of a patient with ornithine transcarbamylase deciency (OTCD).
Another major blow came in 2003, when the Food and Drug Administra-
tion (FDA) placed a temporary halt on all gene therapy trials using retro-
viral vectors in blood stem cells, because children treated with French gene
therapy for X-linked severe combined immunodeciency disease (X-SCID)
had developed a leukemia-like condition. In malignant glioma, on the other
hand, a suicide gene therapy using herpes simplex virus-thymidine kinase
gene/ganciclovir is the rst clinical trial in the world, which started in 1992.
Thereafter, immuno-gene therapies using cytokine genes such as interleukin
or interferon (IFN) were opened one after another. For the past ve years, 7
of 11 clinical trials in USA have included immuno-gene therapies. We have
been also developing a new immuno-gene therapy in Japan. This therapy
using interferon (IFN)- and cationic liposomes is a rst original protocol
developed in Japan. A pilot clinical trial of the therapy began in 2000,
and thereafter the safety and effectiveness were conrmed. In addition,
researchers belonging to Experts on the Gene Therapy Advisory Commit-
tee in the UK have been given approval to carry out a large clinical trial
in 2004, which involves injecting oncolytic herpes simplex virus into the
glioma, because the rst patient to receive the treatment had a long survival
more than 7 years. From this evidence, it is considered that gene therapy
for malignant brain tumors is one of the most promising strategies in all
gene therapies. In this lecture I introduce the current status of gene therapy
research and clinical trials for malignant brain tumors, including ours. In
addition, I comment on the status of gene therapy regulation in Japan, a
new molecular targeting therapy based on the results of genetic analysis in
patients treated with our IFN-gene therapy and the combination of gene
therapy and cell therapy. In the near future I believe that gene therapy can
offer hope to patients with malignant brain tumors, including glioma.
273. THE P75 NEUROTROPHIN RECEPTOR MEDIATES
GLIOMA INVASION
A. Johnston,
1
D. Senger,
2
X. Lun,
2
H. Muzik,
2
Z. Shi,
2
M. Slovack,
2
S.
Robbins,
1
and P. Forsyth
2
;
1
Biochemistry and Molecular Biology and
2
Clinical Neuroscience, University of Calgary, Calgary, Canada
Malignant gliomas are highly invasive brain tumors. This invasive ten-
dency renders these tumors surgically incurable and associates them with
a high mortality rate. Glioma cell invasion is poorly understood, and we
believe that increased understanding of this process will lead to the devel-
opment of novel therapeutics. Through the use of a serial in vivo selection
procedure, we have isolated highly invasive and highly noninvasive cell sub-
populations from the human glioma cell line U87. Microarray comparison
of the two subpopulations was then used to identify novel genes not previ-
ously implicated in glioma invasion. One of the differentially expressed
genes was the p75 neurotrophin receptor. This receptor is present at both
the RNA and protein level in the invasive cell population, but is undetect-
able in the noninvasive population. Importantly, we have also observed
that treatment of these cells with p75 ligands increases the migration and
invasion of the invasive, p75-positive cells, but has no effect on the nonin-
vasive, p75-negative population. In addition, we found that p75 is present in
other glioma cell lines and that a strong positive correlation exists between
levels of p75 expression and neurotrophin-induced migration in these cells.
Subsequently, we upregulated p75 levels in the original U87 cell line and
demonstrated that this upregulation conferred an increased migratory and
invasive ability in vitro. Conversely, downregulation of p75 in a human
glioma cell line expressing high levels of p75 decreased the migration and
invasion of these cells in vitro. Finally, we demonstrated that p75 is overex-
pressed in human glioma patient specimens. These data suggest that p75 is
present, functional, and involved in glioma migration and invasion. Future
experiments are aimed at identifying the downstream signaling molecules
that mediate these effects.
274. HUMAN SKINDERIVED STEM CELLS INHIBIT BRAIN
TUMOR GROWTH
F. Acerbi,
1
C. Giussani,
1
F. Pisati,
2
G. Carrabba,
1
M. Belicchi,
2

V. Lucini,
1
S.M. Gaini,
1
A. Bikfalvi,
3
Y. Torrente,
2
and L. Bello
1
;
1
Neurosurgery and
2
Neurology, University of Milano, Milano, Italy;
3
INSERM Unit EPI 0113, University of Bordeaux 1, Talence, France
Neural stem cells (NSC) implanted into rodent intracranial gliomas
distribute themselves and surround inltrating tumor cells. A number of
questions are left unanswered (the difculty of isolating NSC from adult
tissues, an ethical issue linked to the use of fetal derivatives, and the advan-
tages of NSC in allogenic transplantation). We isolated human skin-derived
stem cells (SDSC) from samples of skin obtained from glioma patients and
studied their behavior in the presence of brain tumors. We initially stud-
ied SDSC targeting ability by implanting SDSC in the contralateral and
homolateral hemisphere of well-established U87 nude mice glioma. SDSC
demonstrated a high tumor target ability. Eight days after injection, SDSC
migrated extensively within the tumor and surrounded the invading tumor
border. In short- and long-term studies, mice injected with SDSC 11 days
after tumor cell implantation showed a decrease in tumor volume and a
longer survival than the controls (50% survival longer of 11 days). Treated
tumors were associated with a decrease in tumor vascularization and inva-
sion in the area of injected SDSC. When SDSC were implanted 5 days after
glioma cell injection, they did not form tumors and resulted in a more sig-
nicant decrease in tumor volume. The ability of SDSC to decrease tumor
vascularization and invasion was conrmed in CAM assay implanted with
U87 tumors. The antitumor effect of SDSC was further studied in tyrp1-Tag
mice that develop retinal pigmented epithelium tumors, rapidly migrating
along the optic nerve and invading the brain. In these mice, intracerebral
SDSC injection decreased the intracranial and extracranial portions of the
tumors. Tumor growth inhibition was associated with a marked decrease
in tumor vascularization and invasion. These data suggest a potential treat-
ment of intracranial tumors by the use of SDSC from the same patients.
275. INFLUENCE OF ANTI-ANGIOGENIC SUBSTANCES
ON NEURAL PROGENITOR CELL MIGRATION,
DIFFERENTIATION, AND TUMOR HOMING
M. Kirsch,
1
O. Heese,
2
G. Schackert,
1
and H. Schackert
1
;
1
Neurosurgery,
Carl Gustav Carus University Hospital Dresden, Dresden;
2
Neurosurgery,
Hamburg-Eppendorf University Hospital, Hamburg; Germany
Neural progenitor cells (NPC) have been used to track solid brain
tumors in vivo as well as to express a therapeutic molecule intratumor-
ally. In addition to their antitumoral properties, a restorative potential
of these NPC is hoped for. We have investigated the use of local expres-
sion of the angiogenic inhibitor endostatin in a model of hematogenous
cerebral melanoma metastases using neural progenitor cells as therapeutic
vehicles. Concomitantly, we have investigated the effects of endostatin and
of additional angiogenesis inhibitors (angiostatin, Su5416, Su5614, and
Cox2-inhibitor) on in vivo migration and differentiation. In a second step,
the therapeutic potential of endostatin-transfected NPCs was investigated.
Migration of NPCs was tested with a modied Boyden-chamber assay and
a modied wounding assay. Endostatin levels were measured by ELISA.
Proliferation and differentiation patterns were analyzed by immunohisto-
chemistry using antibodies against GFAP, NeuN, Huc, MBP, GalC, nestin,
vimentin, and Ki67. The primary NPC from GFP-transgene mice as well as
the myc-immortalized C17.2 NPC line were exposed to murine recombi-
nant endostatin and angiostatin and the tyrosine kinase inhibitors Su5416
and Su5614. The cerebral metastasis model used murine melanoma cells
either injected into the internal carotid artery of mice or placed stereotacti-
cally into the frontal lobe and concomitant injection of NPCs with/without
transgenic endostatin secretion. Survival was recorded, and the brains were
subjected to immunohistochemical analysis. Endostatin and Su5614 led
to a signicant reduction in migration, whereas angiostatin and Su5416
did not alter migration. Phenotypic and proliferative changes compared to
control cells were not observed. Endostatin-transfection of NPCs did not
result in prolonged survival although endogenous endostatin expression by
tumor cells demonstrated survival advantage. Upon inspection of tumors,
a reduced migration of NPCs into metastases was noted if compared to
non-endostatin transfected NPCs. Endostatin inhibited NPC migration.
This observation concurs with the in vivo ndings of reduced NPC homing
toward the tumor and therefore abolishing the therapeutic properties of
NPCs using endostatins anti-angiogenic therapeutic paradigm.
276. ANTIANGIOGENIC AGENT THALIDOMIDE INCREASES
THE ANTITUMOR EFFECT OF SINGLE HIGH-DOSE
IRRADIATION (GAMMA-KNIFE RADIOSURGERY) ON THE
RAT ORTHOTOPIC GLIOMA MODEL
D. Nam and J. Lee; Department of Neurosurgery, Sungkyunkwan
University School of Medicine, Samsung Medical Center, Seoul, Korea
We investigated the effect of gamma-knife radiosurgery (GKR) on a rat
model of glioma with or without simultaneous administration of temozolo-
mide or thalidomide. Combined GKR (20 Gy single maximal dose) and/or
pharmacotherapy (for 3 days including the radiosurgery day) was delivered
on the 18th day after stereotactic implantation of C6 glioma cells. The ani-
mals were sacriced 24 h after GKR for evaluation of apoptosis, PCNA
index, microvessel density, and expression of basic broblastic growth fac-
tor (bFGF) and of vascular endothelial cell derived growth factor (VEGF).
To determine the tumor size reduction, other groups of animals were sac-
riced at 5 days after GKR (with or without pharmacotherapy), which was
delivered 14 days after the tumor inoculation. Compared with the rat which
received GKR alone, there was signicant increase of tumor cell apoptosis
in GKR TMZ as well as GKR thalidomide, signicant decrease of
MVD in GKR thalidomide, and signicant decrease of proliferative index
in GKR TMZ. Five days after the GKR, shrinkage of tumor was most
prominent in the GKR and thalidomide combination group. In addition to
the reduction of bFGF and VEGF expression, reduction of MVD and induc-
tion of apoptosis by thalidomide may be associated with the prominent
shrinkage of tumor among the GKR treatment groups. Our data suggest
that radiosurgery combined with antiangiogenic therapy may be the most
promising combination to control malignant glioma.
277. THE ROLE OF AUTOTAXIN IN GLIOMA INVASION
B. Hoelzinger,
1,2
C. Beaudry,
1
T. Demuth,
1
L. Reavie,
1
L. Mariani,
3
M.
Nakada,
1
and M. Berens
1
;
1
Neuro-genomics, Translational Genomics
Research Institute, Phoenix, Arizona, USA;
2
Arizona State University,
Tempe, Arizona, USA;
3
Department of Neurosurgery, Inselspital, Bern,
Switzerland
One of the salient features of malignant astrocytomas is the propen-
sity to diffusely invade brain parenchyma; it is from these invading cells
that the tumor invariably recurs. A better understanding of the molecular
mechanisms of this locally invasive behavior could yield potential therapeu-
tic targets aimed exclusively at this tumor cell subpopulation. To this end, a
gene expression prole of GBM invasion was created by comparing mRNA
from invasive cells to that of matched noninvasive cells from the tumor core.
Differences in expression patterns between the two laser capture microdis-
sected cell populations revealed multiple genes related to invasion. Among
these was the autocrine motility factor Autotaxin (ATX), which is secreted
by invasive melanoma and as well as by metastatic breast carcinoma and
other malignant tumors. Recently, its role as a lysophospholipase D was
identied, catalyzing lysophosphatidyl choline (LPC) into lysophosphatidic
acid (LPA). LPA stimulates motility in various cell types, including glioma
cells, which express high levels of LPA1 receptor. Evaluation of ATX expres-
sion in a glioma revealed a high expression in the invading cells of astrocytic
tumors of all grades. To investigate the functional role of ATX in astrocy-
toma invasion, stable transfectants carrying wild-type ATX and a catalyti-
cally inactive form (H316Q-U251) were created by using the U251 glioma
cell line. ATX-U251 migrated signicantly faster than H316Q-U251 in a
radial migration assay, an effect that was not reversed by LPA dependent
motility stimulation. Cells expressing high levels of ATX also proved more
adherent to laminin, specically in the presence of its substrate LPC. To
test the property of invasion we used an orthotopic rat brain slice assay,
which showed ATX-U251 to be more highly invasive than H316Q-U251 or
control cells. ATX knockdown with siRNA resulted in lowered invasion of
spheroids in a collagen I matrix, an effect that was reversed with the addi-
tion of LPA. Changes in motility that are induced by LPA receptors result in
cytoskeletal rearrangements brought about by the rho family of GTPases.
We examined the actin cytoskeleton in the poorly motile H316Q-U251 cells
and found normal stress bers, enhanced lopodia formation and reduced
membrane rufes, suggesting modulation of rac signaling. Further studies
consist of elucidating whether the motility inhibition of the secreted protein
H316QATX functions in a paracrine or autocrine manner.
278. INTERPLAY BETWEEN EPHB2 AND EPHRIN-B ON
GLIOMA CELLS PROMOTES GLIOMA MIGRATION AND
INVASION
M. Nakada,
1
J. Niska,
2
W. McDonough,
2
N. Tran,
2
A. Joy,
2
and M.
Berens
2
;
1
Neurology Research, Barrow Neurological Institute, Phoenix,
Arizona;
2
The Translational Genomics Research Institute, Phoenix,
Arizona; USA
The receptor tyrosine kinases of the EphB family and their ephrin-B
ligands play a key role in neurodevelopmental processes such as boundary
formation, vasculogenesis, and cell migration. The Eph receptors trans-
mit forward signals via their kinase domain and reverse signals via their
transmembrane ephrin-B ligands. EphB2 are upregulated in glioblastoma,
especially in invading glioma cells (Nakada et al., Cancer Res. 64, 2004).
Here we use a soluble EphB2 ectodomain fusion protein (EphB2/Fc) to dem-
onstrate that ephrin-B transduces signals that regulate cell migration and
invasion. EphB2/Fc induced ephrin-B tyrosine phosphorylation, migration,
and invasion in in vitro and in ex vivo rat brain slice model using U87 and
U251 glioma cells. Confocal imaging showed ephrin-B localized in lamel-
lopodia of motile U87 and U251 cells. Activation of ephrin-B by EphB2/Fc
induced phosphorylation of Akt. The phosphatidylinositol 3-kinase (PI3-K)
inhibitor LY294002 reduced EphB2/Fc stimulated migration and invasion
concomitant with phosphorylation of Akt. Human brain tumor specimens
portrayed higher expression of ephrin-B in glioblastoma than in low-grade
astrocytomas or normal brain. Immunohistochemistry showed phosphory-
lated form of ephrin-B localization primarily in glioblastoma cells. Taken
together, ephrin-B stimulated by EphB2 transduces outside-in signals that
affect migration and invasion in glioma through the PI3-K/Akt pathway.
This study was supported by the American Brain Tumor Association (MN),
NS042262 and NS043446.
279. HIF-1A EXPRESSION IS ASSOCIATED WITH GLIOMA
CELL INVASION
T. Demuth,
1
D. Hoelzinger,
1
J. Rennert,
1
E. Newcomb,
2
M. Berens,
1
and
D. Zagzag
2
;
1
Neurogenomics Department, Brain Tumor Unit, TGen,
Phoenix, Arizona;
2
Department of Pathology, NYU Medical School, New
York, New York;
3
Arizona State University, Phoenix, Arizona; USA
Hypoxia is associated with adverse outcome for a number of solid
tumors, including gliomas. We reported expression of hypoxia inducible
factor-1a (HIF-1a) in two different microenvironmental areas of gliomas:
(1) pseudopalisading cells at the tumor core, and (2) invasive tumor cells
located in brain adjacent to tumor (BAT). Using CA9, a carbonic anhydrase,
as a surrogate marker for tumor hypoxia, we showed by immunohistochem-
istry (IHC) that HIF-1a expression within these two brain tumor areas
may be driven by different mechanisms (possibly leading to distinct cellular
responses): Hypoxic cells within the pseudopalisades exhibited staining for
HIF-1a and CA9, whereas all the invading HIF-1a positive glioma cells in
the BAT were negative for CA9. These results suggest that upregulation of
HIF-1a in invading glioma cells assessed by IHC might be controlled by
molecular mechanism(s) other than those induced by hypoxia. The asso-
ciation of HIF-1a expression with glioma invasion was further studied by
comparing the gene expression prole of invasive and stationary glioma
cells isolated from human biopsies by laser capture microdissection. In two
of three tumor samples HIF-1a was found to be upregulated in invasive
cells while VEGF, a gene known to be induced by HIF-1a under hypoxic
conditions, was downregulated. Transcriptional regulation of HIF-1a was
also examined by microarray analysis of in vitro migration assays. Three
of ten GBM cell lines induced to migrate on glioma-derived extracellular
matrix showed HIF-1a upregulation. Gene expression changes coincident
with upregulation of HIF-1a and glioma invasion will be reported by com-
paring in vivo transcriptomes from human glioma biopsy specimens with
those obtained from in vitro invasion assays. These data and the results
from IHC for CA9 and HIF-1a suggest that HIF-1a may be activated and
signal through a distinct and separate pathway unique for glioma cell inva-
sion. We propose dual modes of activated expression leading to detection
of HIF1a-protein in invasive and stationary glioma cells by IHC: (1) In
the hypoxic environment of tumor core HIF-1a is stabilized and activates
transcription of hypoxia induced genes; (2) in glioma cells at the invasive
edge, increased transcription of HIF-1a may compensate for its ubiquitina-
tion/degradation under normoxic conditions. Disparate drivers of HIF-1a
expression in glioma cells in response to microenvironental cues may point
to exploitable targets in the invasive cells.
280. THE SOLUBLE DECOY RECEPTOR FOR VEGF, VEFG
TRAP, INDUCES ANTIGLIOMA EFFECT IN VIVO
C. Gomez-Manzano,
1
J. Holash,
4
J. Xu,
1
O. Lee,
1
J. Fueyo,
1
J. Rudge,
4

X. Zhou,
2
B.N. Bekele,
2
K. Aldape,
3
and W.K.A. Yung
1
;
1
Neuro-
Oncology,
2
Biostatistics, and
3
Pathology, The University of Texas M.D.
4
Regeneron Pharmaceuticals,
Inc., Tarrytown, New York; USA
Pathological angiogenesis is a hallmark of cancer, and specically of
glioblastomas, the most malignant and common form of primary brain
tumors. VEGF is the key molecular player involved in the mechanism of vas-
cular growth. We undertook this project to test VEGF Trap, a new antian-
giogenic agent that acts as a soluble decoy receptor for VEGF. This molecule
incorporates domains of both VEGFR-1 and VEGFR-2 and binds VEGF
with high afnity. U-87 MG human glioma cells were implanted in the
brain of nude mice, and VEGF Trap was administered (25 mg/kg sc, twice
a week for a total of 3 weeks) at days 0, 4, and 10 after cell implantation.
hFc and PBS were used as control treatments. Serum was collected three
days after the initial dose to assess circulating levels of VEGF Trap. Serial
temporal examination of the brains of untreated mice showed that tumors
grew to a volume of 0.3 mm
3
and exhibited a very low microvascular den-
sity (MVD 6 vessels/0.5 mm
2
), with central necrosis within four days of
implantation, and peripheral reactive vasculature. At that time point, treat-
ment of glioma-bearing animals with VEGF Trap increases signicantly the
survival time of animals compared to that of control-treated animals (P
0.007, log-rank test). Another group of animals was treated starting day 10
of the experiment, at the time that tumors were masses of cells with volumes
of 3045 mm
3
and had fully developed vasculature (MVD 3035 ves-
sels/0.5 mm
2
). At this stage of the disease, VEGF Trap treatment induced
a signicant prolongation of animal survival (P 0.0001, log-rank test).
VEGF Trap was detected in the serum of the animals at levels of about 50
g/ml or greater. Taken collectively, our data indicate that treatment with
VEGF Trap results in prolongation of survival in this intracranial human
glioma xenograft model.
281. DOMINANT-NEGATIVE INHIBITION OF THE
RECEPTOR TYROSINE KINASE RECEPTOR AXL
SUPPRESSES GLIOMA CELL MIGRATION AND INVASION
AND PROLONGS SURVIVAL
P. Vajkoczy,
1
A. Kunkel,
1
R. Erber,
1
H. Capelle,
1
U. Eichelsbacher,
2
and
A. Ullrich
2
;
1
Department of Neurosurgery, Klinikum Mannheim der
Universitat Heidelberg, Mannheim;
2
Department of Molecular Biology,
Max-Planck-Institute for Biochemistry, Martinsried; Germany
Receptor tyrosine kinases (RTKs) play an important role in growth
and progression of brain tumors. Our previous work has shown that the
RTK AXL, whose biological function has remained obscure so far, is over-
expressed by glioma cell lines and human astroglial tumors. The aim of
the present study was to analyze the role of AXL in glioma biology. Two
glioma cell lines, one expressing high levels of AXL (i.e., SF126) and the
other lacking intrinsic AXL (i.e., SF767) were transfected to overexpress
either the human wild-type form (AXL-WT) or a truncated, dominant-
negative mutant form (AXL-DN). Glioma cell morphology and cell behav-
ior with respect to proliferation, aggregability, migration, and invasion
were assessed in vitro. To study the relevance of AXL for tumor growth, the
glioma cell lines were implanted subcutaneously and into the brains of nude
mice. Finally, glioma cells were implanted into the dorsal skinfold chamber
model to assess tumor cell behavior, tumor angiogenesis, and tumor perfu-
sion in vivo by intravital multi-uorescence microscopy. SF126-AXL-DN
cells were characterized by reduced cell-to-cell contacts, a moderately
reduced proliferative activity, and most importantly by a severe impair-
ment of tumor cell migration and tumor cell invasion. In vivo, subcutane-
ous SF126-AXL-DN tumor growth was reduced by 97%, and tumor cell
invasion into the adjacent tissue was suppressed. Finally, survival following
intracerebral implantation of SF126-AXL-DN cells was signicantly pro-
longed compared to SF126-AXL-WT cells. In contrast, inhibition of AXL
signaling in SF767 cells had no signicant effects. Our study provides the
rst evidence that the tyrosine kinase receptor AXL modulates migration
and invasion of human glioma cells and that inhibition of AXL signaling
suppresses tumor expansion and prolongs survival by blocking tumor cell
invasion. Thus, AXL may represent a novel molecular target for the treat-
ment of malignant glioma.
282. INHIBITION OF GLIOBLASTOMA ANGIOGENESIS
AND INVASION BY COMBINED TREATMENTS DIRECTED
AGAINST VEGFR-2, EGFR AND VE-CADHERIN
K. Lamszus,
1
M.A. Brockmann,
1
C. Eckerich,
1
P. Bohlen,
2
C. May,
2
R.
Fillbrandt,
1
and M. Westphal
1
;
1
Department of Neurosurgery, University
2
ImClone Systems,
New York, New York, USA
Increasing evidence suggests that inhibition of tumor angiogenesis can
inuence tumor cell invasion and metastasis. We previously showed that
systemic antagonization of vascular endothelial growth factor receptor-2
(VEGFR-2) with the monoclonal antibody (mAb) DC101 inhibited glio-
blastoma growth in an orthotopic model, but caused increased tumor cell
invasion along the preexistent vasculature. In human glioblastoma cells,
signaling through the epidermal growth factor receptor (EGFR) predomi-
nantly stimulates tumor cell invasion. Therefore, we attempted to inhibit
tumor cell invasion caused by DC101 therapy by combined systemic treat-
ment with a mAb against EGFR (C225). In addition, we analyzed whether
antagonization of vascular endothelial (VE)-cadherin as a different anti-
angiogenic target can also inhibit glioblastoma growth and whether this
also stimulates invasion. Treatments were either initiated on day 1 after
intracerebral tumor cell injection or on day 6 when tumors were already
established. Increased tumor cell invasion caused by DC101 monotherapy
was inhibited by 50%66% through combined treatment with C225 and
DC101. C225 inhibited glioblastoma cell migration in vitro, but had no
effect on the volume of the main tumor mass or on tumor cell prolifera-
tion or apoptosis in vivo, neither alone nor in combination with DC101.
The anti-VE-cadherin mAb E4G10 also inhibited tumor angiogenesis
and growth, although with weaker effects than DC101, and the effects of
E4G10 were dependent on early initiation of treatment. E4G10 treatment
caused increased tumor cell invasion along the host vasculature, although
also with weaker effects than DC101. Our ndings show that anti-angio-
genic glioblastoma therapy targeting either VEGFR-2 or VE-cadherin can
inhibit tumor growth, but can increase tumor cell invasion in an orthotopic
model. The increased tumor cell invasion caused by DC101 treatment can
be inhibited by simultaneous antagonization of EGFR, which in the context
of human glioblastomas has been implicated in tumor cell invasion.
283. CONTACTIN IS EXPRESSED IN HUMAN ASTROCYTIC
GLIOMAS AND MEDIATES REPULSIVE EFFECTS
C. Eckerich,
1
S. Zapf,
1
U. Ulbricht,
1
S. Mller,
2
R. Fillbrandt,
1
M.
Westphal,
1
and K. Lamszus
1
;
1
2
AGY Therapeutics,
Inc., San Francisco, California, USA
Using subtractive cloning, we identied contactin as overexpressed in
glioblastomas compared with normal brain. Contactin is a cell surface adhe-
sion molecule that is normally not expressed by astrocytes. It is expressed
by neurons and oligodendrocytes at particularly high levels during develop-
ment. Contactin can mediate adhesive or repulsive intercellular interactions
depending on the molecular context. We analyzed the expression of con-
tactin in human astrocytomas and determined its functional relevance for
glioma cells. Western blotting, immunohistochemistry and confocal immu-
nocytochemistry were used to analyze contactin expression in astrocytomas
and astrocytes. Adhesion and migration assays were performed to study
effects of contactin on glioma cells. Contactin cDNA was transfected into
glioma cells to determine the effect of contactin overexpression on attach-
ment to extracellular matrix (ECM) molecules. Expression of the receptor-
type protein tyrosine phosphatase (RPTP), a specic contactin ligand that
is also overexpressed in glioblastomas, was downregulated by stable siRNA
transfection to study interactions with contactin overexpressing cells.
Contactin expression was detected in GFAP positive tumor cells but was
absent in normal astrocytes. Levels of contactin in gliomas were associ-
ated with increasing malignancy grade. None of 10 glioblastoma cell lines
adhered to contactin or was stimulated in its motility by contactin. In con-
trast, increasing coating concentrations of contactin caused progressive cell
repellence. Co-presentation with contactin reduced the haptotactic migra-
tion induced by bronectin in several cell lines. Overexpression of contactin
in human glioblastoma cells by cDNA transfection had no effect on cell
proliferation or adhesion to various ECM molecules. Contactin expression
also did not alter the adhesion to cells expressing normal or downregulated
levels of RPTP. The expression of astrocytic as well as neuronal markers
within glioma cells may reect an ability of these cells for multilineage dif-
ferentiation, a phenomenon that has recently been described also for the
so-called glioma stem cells. While adhesive and proliferative properties of
glioma cells are unaltered by contactin overexpression, confrontation of
glioma cells with contactin has repulsive effects, which may contribute to
the diffuse inltration pattern characteristic of these cells in human brain.
284. INHIBITION OF GLIOBLASTOMA GROWTH BY SIRNA-
MEDIATED ANTAGONIZATION OF RPTP
U. Ulbricht, C. Eckerich, R. Fillbrandt, M. Westphal, and K. Lamszus;
Department of Neurosurgery, University Hospital Hamburg-Eppendorf,
Hamburg, Germany
We previously identied the receptor-type protein tyrosine phosphatase
(RPTP) and its ligand pleiotrophin (PTN) as overexpressed in glioblasto-
mas using cDNA arrays (Mller et al., Oncogene, 2003). Both molecules
have been implicated in neuronal migration during central nervous system
development. We conrmed RPTP and PTN overexpression in glioblasto-
mas at the protein level and showed that matrix-immobilized PTN stimu-
lates glioma cell migration (Ulbricht et al., J. Neuropathol. Exp. Neurol.,
2003). In the present study, we analyzed the effect of RPTP expression
on glioma growth in vitro and in vivo using siRNA technology. RPTP
expression was downregulated in U251-MG glioblastoma cells by stable
siRNA transfection. Downregulation was detected by Northern and West-
ern blotting. Colorimetric proliferation assays and modied Boyden cham-
ber haptotactic migration assays were performed in vitro. Tumor growth
in vivo was studied using a subcutaneous nude mouse model. Tumors were
analyzed immunohistochemically to assess cell proliferation, apoptosis and
vascularization. Two siRNA transfected clones with strong downregulation
of RPTP expression (RPTP) were obtained, and these clones were sub-
sequently compared with 2 mock transfected control clones (RPTP). Pro-
liferation of RPTP clones was inhibited by 56% to 90% compared with
RPTP clones. Haptotaxis (migration toward substrate-bound molecules)
induced by PTN was inhibited by 52% to 76% in RPTP clones compared
with controls. Collagen I and bronectin were also haptotactic. However,
no differences were observed between RPTP and RPTP clones, sug-
gesting that the inhibition of haptotaxis toward PTN is specically medi-
ated through RPTP. In vivo, growth of RPTP clones was inhibited by
93% to 98% compared with controls. The fraction of proliferating tumor
cells was reduced by 49% to 74% in tumors derived from RPTP clones,
whereas no differences were observed for apoptosis or microvessel den-
sity. Glioma growth in vivo can be inhibited by downregulation of RPTP
expression. In vitro, RPTP contributes to glioma cell proliferation and
mediates PTN-induced haptotactic migration. PTN was demonstrated ear-
lier to be secreted by U251-MG cells. Therefore, our ndings suggest that
upregulated expression of PTN and RPTP in human astrocytoma cells can
create an autocrine loop that contributes to tumor growth in vivo.
285. REGULATION OF PATHOLOGICAL VASCULATURE OF
MALIGNANT ASTROCYTOMAS BY ANGIOPOIETIN-1
G. Zadeh, R. Reti, K. Koushan, Q. Baoping, P. Shannon, and A. Guha;
Neurosurgery Division, Toronto Western Hospital, Toronto, Canada
A histopathological hallmark of malignant astrocytomas is microvas-
cular proliferation and formation of vascular entities referred to as glo-
meruloid bodies. The signicance of glomeruloid bodies and the molecular
mechanisms driving the abnormal vascular architecture in human malig-
nant astrocytomas are not known. Vascular endothelial growth factor-A
(VEGF-A) is known to be the main angiogenic regulator of tumor angiogen-
esis in malignant astrocytomas. However, a direct link between VEGF-A
and generation of glomeruloid bodies in tumor models, in particular astro-
cytomas, has not been established. Angiopoietins (Ang1 and Ang2) and
their endothelial-cell (EC)-specic receptor-tyrosine-kinase Tie2, have also
been implicated as important regulators of both normal and pathological
tumor angiogenesis. In this study we have focused on the potential role of
Ang1 as a regulator of glomeruloid bodies in malignant astrocytomas. U87
human GBM cell lines with constitutive or Tet-Off regulated overexpression
of Ang1 and vector controls in subcutaneous or stereotactic intracranial
xenograft models in Nod-Scid mice were used. Tie2 inhibition of the tumor
vasculature was undertaken with a kinase-dead dominant-negative Tie2,
containing only the extracellular domain, termed ExTek. Ex-Tek was puri-
ed from a baculovirus expression system, and intratumoral injections with
a screw-guided system were undertaken. At necropsy, FactorVIII staining
was undertaken to visualize the tumor microvasculature and computer-
assisted image guided analysis undertaken. Overexpression of Ang1 by
human malignant astrocytoma cell lines in subcutaneous or intracranial
xenografts reproduce many of the vascular features of human malignant
astrocytomas, including glomeruloid bodies. The association of glomeruloid
bodies was dependent on Tet regulated levels of Ang1 expressed and was
inhibited by blocking the Tie2 receptor by ExTek. Furthermore, although
Ang1 mediated astrocytoma angiogenesis and growth also requires VEGF-
A, induction of glomeruloid bodies by Ang1 was independent of VEGF-A.
These results suggest that the pathological piling of EC and generation of
glomeruloid bodies is regulated by Ang1.
286. IMINO SUGAR-BASED GANGLIOSIDE INHIBITORS
DOWNREGULATE INVASIVE BEHAVIOR IN MALIGNANT
GLIOMA IN VITRO
G. Pilkington, Y. Kyriazis, Y. Orphanos, M. Christidou, V. Drewett, and
D. Schelshorn; Cellular and Molecular Neuro-oncology Group, School of
Pharmacy and Biomedical Sciences, University of Portsmouth, UK
Gangliosides are acidic glyco-sphingolipids characterized by the pres-
ence of one or more sialic acid residues. In neoplastic tissues simple ganglio-
sides are overexpressed and their role in tumor-tumor cell interactions as
well as the interactions between healthy cells and tumor cells is pivotal to
tumor growth, development, and metastasis/invasion. Simple gangliosides
(e.g., GM3 and GD3) modulate neoplastic cell adhesion as well as produc-
tion of extracellular matrices such as laminin as well as certain degradative
enzymes (metalloproteinases). The function of integrins, integral compo-
nents of the cell-to-cell adhesion process, is also modulated by gangliosides.
A crucial step in ganglioside biosynthesis is the glucosylation of the ceramide
moiety that leads to the formation of glucosylceramide, the backbone of
gangliosides. This reaction is catalyzed by glucosylceramide synthase, inhi-
bition of which by the use of the imino sugar, N-butyl-deoxynojirimycin
(NB-DNJ), and other structurally similar compounds (such as NB-DGJ),
result in reduced or ablated ganglioside synthesis. By use of a highly inva-
sive anaplastic astrocytoma culture (IPSB-18) and a cultured (IPTP) giant
cell variant glioblastoma (a tumor which, although histologically similar to
classical glioblastoma, shows a limited degree of inltrative behavior, both
in vivo and in vitro), we have examined the modulatory effect of 4 imino
sugars with structural similarities to NB-DGJ on ganglioside expression
(immunocytochemistry, ow cytometry, and HPLC) as well as on inva-
sive behavior in glioma by the use of time-lapse microscopy and Transwell
modied Boyden chamber invasion assays. We also assessed MMP-2 and -9
and TIMP gene expression (TaqMan RT-PCR) before and after treatment.
All agents inhibited ganglioside expression (in particular GD3), albeit to
different extents. Moreover, a differential downregulatory effect was seen
on expression of MMPs/TIMPs as well as on invasion. Results were more
marked in the invasive IPSB-18 line than in IPTP. These imino sugar agents
not only show that targeting of ganglioside synthesis pathways may prove
of benet in reduction of glioma invasion but also may have a potential role
to play in clinical neuro-oncology. This work was supported, in part, by
Oxford Glycosciences.
287. ANGIOSUPPRESSIVE THERAPY TARGETING HIF-1/
VEGF FOR MALIGNANT GLIOMAS IN VITRO AND IN VIVO
S. Takano, H. Kamiyama, K. Tsuboi, and A. Matsumura; Department
of Neurosurgery, Institute of Clinical Medicine, University of Tsukuba,
Tsukuba City, Japan
Hypoxia inducible factor-1 (HIF-1) is an important molecular target
because it is a mediator of hypoxic condition, VEGF induction, and radia-
tion/chemo resistance. We evaluated angiosuppressive action of CPT-11,
YC-1, and 2ME (2-methoxyestradiol) for ACNU resistant gliomas. In vitro,
active metabolite SN38, YC-1, and 2ME were evaluated for (1) endothelial
cell proliferation with WST assay, (2) vascular tube formation with angio-
genesis kit (Kurabo), and (3) VEGF and hypoxia inducible factor 1a (HIF1a)
expression of U87 malignant glioma cells with quantitative RT-PCR, West-
ern blot, and ELISA. In vivo, CPT-11 low (10 mg/kg) and high (40 mg/
kg) dose was evaluated for (1) U87 subcutaneous growth and survival and
(2) ACNU resistant U87 subcutaneous growth with immunohistochemi-
cal evaluation of angiogenesis (VEGF expression, vessel density, MIB-1,
apoptotic index). In vitro, SN38 effectively inhibited the endothelial cell
proliferation and vascular tube formation at non-toxic concentrations (less
than 0.01 M). Also, SN38 decreased HIF-1a and VEGF expression of
glioma cells in a dose- and time-dependent manner under hypoxic condi-
tion. Although 2ME inhibited the HIF-1 and VEGF expression, the inhibi-
tory concentration was more than 25 M. In vivo, CPT-11 even at low dose
inhibited malignant glioma growth, inhibiting tumor cell proliferation and
enhancing tumor cell apoptosis, in addition inhibiting angiogenesis, and
decreased number of tumor vessels and decreased expression of VEGF, a
most important angiogenic factor of gliomas. Based on the angiosuppres-
sive effect of CPT-11, the metronomic and angiosuppressive scheduled CPT-
11 chemotherapy may be promising for malignant glioma treatment.
288. FUNCTIONAL AND PHENOTYPICAL
CHARACTERIZATION OF ENDOTHELIAL CELLS DERIVED
FROM GLIOMAS OF DIFFERENT GRADES
S. Miebach, S. Grau, O. Schnell, S. Schichor, J. Tonn, and R.
Goldbrunner; Neurosurgery, Universiteitsklinikum Grosshadern,
Munchen, Germany
The ability to induce neo-angiogenesis is a key feature in glioma grad-
ing. Investigations of endothelial cells (EC) from various organs revealed
signicant differences between the populations depending on organ and
dignity of the tissue of origin. For assessment of tumor-induced angio-
genesis, use of organ- and tissue-specic EC appears mandatory. EC were
isolated from 22 low-grade astrocytomas (LGEC), 11 malignant gliomas
(HGEC), and 7 normal brain specimens (obtained from epilepsy surgery
procedures). Isolation was performed by combining density gradient sepa-
ration with magnetic bead sorting. Characterization of EC was performed
by measuring the expression of EC-specic cell surface antigens. On mRNA
level expression of MMP 2, 3, 7, 9 and TIMP 1, 2 and 3 was measured by
real-time rt-PCR. Activity of MMP 2 and 9 was assessed by zymography.
Functional ability of the cells was evaluated by means of proliferation, tube
formation, and co-culture. A reproducible method for isolating EC from
brain tumors could be established. There was no correlation seen between
antigen expression pattern and cell morphology. Expression of EC-specic
antigens was strongly inuenced by cell culture conditions; surface antigens
like Glut-1 and wWF were expressed only in primary cultures, while CD31
and VE-Cadherin could be shown present up to passage ve. Regarding
proliferation, there were major differences between EC subpopulations,
with HGEC showing a fourfold higher proliferation rate than LGEC and
NBEC. In MMP expression, mRNA for MMP 2, 3, and 7 was found in all
EC; MMP 9, however, was exclusively expressed in HGEC. None of the EC
isolated could form tubes in vitro, even in presence of various stimulating
factors. Due to rapid changes in EC antigen expression the use of freshly iso-
lated cells in very low passages should be obligatory. Some rst functional
assessments show clear differences between NBEC, LGEC, and HGEC,
pointing toward a different biological behavior among these cell popula-
tions. Thus, for investigating glioma angiogenesis the use of glioma specic
EC is mandatory. The EC differences shown above may reveal a contribu-
tion of endothelial cells to the malignant phenotype of the tumor.
289. NOVEL PROBES FOR MOLECULAR MAGNETIC
RESONANCE IMAGING (MMRI) OF GLIOMAS
B.J. Bedell,
1
S. Assadian,
2
S. Mzengeza,
3
R.F. Del Maestro,
4
and A.C.
Evans
3
;
1
Department of Pathology,
2
Brain Tumor Research Centre,
3

Department of Neurology & Neurosurgery,
4
Brain Tumor Research
Centre and Department of Neurology & Neurosurgery, McConnell Brain
Imaging Centre, Montreal Neurological Institute, Montreal, Canada
Numerous factors, including hypoxia-inducible factor-1a (HIF-1a), vas-
cular endothelial growth factor (VEGF), and matrix metalloproteinases
(MMPs), are strongly associated with tumor angiogenesis and invasion.
These proteins have become a central focus of novel targeted therapies.
The current methods of evaluating the expression of these targets in tissue
specimens, however, do not allow for a complete spatio-temporal picture
of the changes occurring in the tumor. The ability to noninvasively monitor
the efcacy of therapeutic intervention on the expression/activity of these
molecular targets would greatly facilitate drug development, shortening the
time from the bench to clinical use. The advantages of MRI over other
modalities has made it a promising candidate for the imaging of molecular
targets. The emergence of molecular MRI (mMRI), however, has been lag-
ging primarily due to the low detectability of conventional MRI contrast
agents. We have overcome this limitation by developing novel probes which
modulate microvascular permeability, providing a sensitivity much higher
than conventional paramagnetic contrast agents. Quantitative analyses of
changes in local microvascular permeability following administration of
a targeted probe provide an estimation of the relative tissue content of the
molecular entity of interest. These probes were investigated for their abil-
ity to detect the expression of enzymes non-invasively, in vivo in a rat C6
glioma model. C6 glioma cells were transfected with a secreted lacZ gene,
which allows for the secretion of -galactosidase into the tumor interstitium,
as a model tumor-localized enzyme system. Spheroids of the transfected
cell line were implanted into cerebral hemispheres of male Sprague-Dawley
rats. Animals implanted with nontransfected spheroids served as the control
group. Two weeks post-implantation, the animals were imaged using a Sie-
mens 1.5 T whole-body MRI scanner. T1-weighted images were acquired
following intravenous administration of probe, and quantitative maps
of enzyme expression were generated. Following completion of imaging,
enzyme expression was determined on tissue sections. Quantitative MR
maps successfully demonstrated enzyme expression within the transfected
tumors, but not the control tumors. The image data was correlated with
expression of the enzyme on tissue sections. These studies also demon-
strated the ability of these probes to detect temporal changes in the molecu-
lar mediators of angiogenesis. These novel probes promise the successful
MR imaging of the expression of various molecular targets localized to
gliomas in vivo and should nd numerous applications, including the evalu-
ation of novel, targeted therapeutic agents.
290. TUMOR-PRODUCED EXTRACELLULAR MATRIX
INFLUENCES BRAIN TUMOR TROPISM OF HUMAN
NEURAL STEM CELLS
N.O. Schmidt,
1
M. Ziu,
2
T. Cargioli,
2
K. Aboody,
3
P. Black,
2
and R.
Carroll
2
;
1
Department of Neurosurgery, University Hospital Hamburg-
Eppendorf, Hamburg, Germany;
2
Neurosurgical Oncology Laboratory,
Neurosurgery, Brigham & Womens Hospital, Boston, Massachusetts,
USA;
3
Divisions of Hematology/HCT, City of Hope National Medical
Center and Beckman Research Institute, Duarte, California, USA
A major obstacle in the treatment of gliomas is the invasive capacity of
the tumor cells. Previous studies have demonstrated the capability of neural
stem cells (NSCs) to target inltrated tumor cells and to deliver therapeutic
gene products. However, the signals involved in the brain tumor tropism
of NSCs and their interactions within the tumor environment are hardly
dened. As gliomas progress and invade, an extensive modulation of the
extracellular matrix (ECM) occurs. Tumor-ECM derived from 6 glioblas-
toma cell lines and puried ECM compounds known to be upregulated
in the glioma environment were analyzed for their effects on NSC motil-
ity in vitro. We found that tumor-produced ECM was highly permissive
for NSC migration. Laminin was the most permissive substrate for human
NSCs migration, and tenascin-C the strongest inducer of a directed human
NSC migration (haptotaxis). A positive correlation between the degree of
adhesion and migration of NSCs on different ECM compounds exists, as
for glioma cells. Our data indicate that the ECM of malignant gliomas
is a signicant modulator of NSC migration. ECM proteins preferentially
expressed in areas of glioma cell invasion may serve as additional local
guidance signal for NSC tropism to disseminated tumor cells.
291. INHIBITION OF HIF-1A BY SHORT HAIRPIN RNA
TECHNOLOGY INHIBITS BOTH IN VITRO AND IN VIVO
HUMAN GLIOMA CELL GROWTH
R. Jensen and D. Gillespie; Neurosurgery, Huntsman Cancer Institute,
Salt Lake City, Utah, USA
Hypoxia-inducible factor-1a (HIF-1a) is the major regulator of vascular
endothelial growth factor (VEGF). VEGF is thought to be the principal
mediator of peritumoral edema and angiogenesis in malignant gliomas.
Interruption of VEGF secretion could result in growth inhibition of these
tumors. We examine the role of inhibition of HIF-1a using short hairpin
RNA (shRNA) techniques in the growth of human gliomas. Malignant
glioma cell lines were stably transfected with vectors expressing either
shRNA directed against the HIF-1 gene or with control plasmids containing
nonsense shRNAs. HIF-1 and VEGF expression was examined by immu-
nohistochemistry, Western blot, and RT-PCR. In vitro and in vivo growth
studies were carried out on these cells. Measures of proliferation, angio-
genesis, tissue perfusion, and hypoxia were performed on cells in culture as
well as on xenograft tumors. HIF-1a siRNA transfected cells demonstrate
inhibition of both HIF-1a expression and VEGF secretion. In vitro growth
is slightly decreased by shRNA directed toward HIF-1a, while vivo growth
is signicantly decreased in these cells compared to control cells. Interest-
ingly, the plasmid expressing the anti-HIF-1 shRNA is still present in the
xenograft cell after 75 days of implantation in the mouse. Cell prolifera-
tion and angiogenesis are decreased in the tumors containing the shRNA
inhibiting HIF-1. Preliminary results of intratumoral and intravenous use
of anti HIF-1 siRNAs in established xenograft human gliomas is discussed.
Inhibition of HIF-1a results in inhibition of VEGF secretion, glioma tumor
growth and angiogenesis. Further studies are necessary, but this study sug-
gests potential clinical applications of targeting HIF-1a mediated growth
for inhibition of glioma growth and angiogenesis.
292. TWIST: A NOVEL CANDIDATE OF CELL INVASION
REGULATOR IN MALIGNANT GLIOMA
M. Noha
1
, M. Nakane
1
, M. Hirata
1
, H. Nakayama
1
, D. Yoshida
2
, and
A. Teramoto
2
;
1
Department of Neurosurgery, Teikyo University School
of Medicine, Kawasaki;
2
Department of Neurosurgery, Nippon Medical
School, Tokyo; Japan
Malignant glioma is hallmarked as one of the most invasive tumors in
the human. According to recent literature, 18 months would be the best
long-term median survival. Twist, which has been noted to play an essen-
tial role on carcinoma metastasis, expresses in malignant glioma cell lines
also. However, that function is unclear. We hypothesized and investigated
an interaction between TWIST and the invasiveness of malignant glioma.
Malignant glioma cell lines U87MG and U251MG, which have already
conrmed TWIST expression, were employed to evaluate the inhibitory
effect on cell invasion after silence of the TWIST gene using specic siRNA,
with zymogram and chemotaxis assay. In the zymogram assay, MMP-2 and
MMP-9 were suppressed after gene silence. Furthermore, under the same
conditions, the numbers of invading cells through Matrigel were signi-
cantly reduced, as compared with control cells using non-sense siRNA. In
several recent reports, TWIST is highlighted as associated with metastasis
of breast cancer and other malignant tumors. We reveal here that TWIST
could associate with the cell invasion in malignant glioma through MMP-2
and MMP-9 expression, which is reported for the rst time. This might
pioneer a new horizon on the invasiveness of malignant glioma.
293. DOWNREGULATION OF ADAMTS-8 (METH-2) IN
GLIOMAS AND OTHER BRAIN TUMORS
J. Dunn,
1
J. Reed,
1
D. du Plessis,
2
E. Shaw,
1
P. Reeves,
2
A. Gee,
1
P.
Warnke,
2
and C. Walker
1
;
1
JK Douglas Laboratory, Clatterbridge Cancer
Research Trust, Bebington;
2
Department of Neurological Science,
University of Liverpool, Liverpool; UK
ADAMTS-8 (Meth-2) is a member of the ADAMTS (a disintegrin
and metalloproteinase with thrombospondin motifs) family of proteases
comprising 19 genes. ADAMTS-8 is a member of the family subgroup of
aggrecanases (ADAMTS-1, -4, -5, -8 and -15) that are able to cleave the
matrix proteoglycan aggrecan. Furthermore, ADAMTS-1 and ADAMTS-
8 also have potent anti-angiogenic properties. ADAMTS-8 expression
has been shown to be downregulated in breast carcinoma and NSCLC.
In lung tumors this gene may be silenced by promoter hypermethylation.
ADAMTS-8 has not yet been studied in brain tumors. Therefore, the aim
of our study was to determine the importance of ADAMTS-8 in the patho-
genesis of gliomas and other brain tumors. Expression of ADAMTS-8 in
normal whole brain, cerebral cortex, frontal lobe, cerebellum, meninges
and lung, 38 brain tumors (22 gliomas, 4 meningiomas, 7 metastastic car-
cinomas, 1 hemangioblastoma and 1 medulloblastoma) and 4 glioma cell
lines (U373, Hs683, T98G and U87MG) was studied by using quantitative
RT-PCR. The expression of the anti-angiogenic TSP-1 and the angiogenic
VEGF was also investigated in a subset of 28 of the original 38 tumors.
Promoter hypermethylation analysis of ADAMTS-8 by Methylation Spe-
cic-PCR was undertaken in 31 brain tumors, two normal brain samples,
and the aforementioned four glioma cell lines. ADAMTS-8 was highly
expressed at similar levels in normal whole brain, cerebral cortex, frontal
lobe, cerebellum, meninges, and lung. Furthermore, analysis of ADAMTS-
8 in the brain tumors and glioma cell lines indicated a decrease in mRNA
levels of at least twofold in comparison to normal whole brain in all tumors
and cell lines, and no detectable ADAMTS-8 transcript in 18/38 tumors.
In contrast to ADAMTS-8, expression of TSP-1 was upregulated in the
majority of tumors (54%), while VEGF was upregulated in less than half
(43%). There was no correlation between the expression patterns of these
genes. One metastatic carcinoma and three cell lines (U373, T98G and
U87MG) showed hypermethylation of the ADAMTS-8 promoter region.
These data suggest that downregulation of ADAMTS-8 may be important
in the pathogenesis of a range of brain tumor types, whereas the regulation
of expression of TSP-1 and VEGF may be more variable. Our results also
suggest that an alternative, and as yet unknown, mechanism of silencing of
ADAMTS-8 occurs in the majority of the brain tumors tested.
294. INTER ALPHA TRYPSIN INHIBITOR HEAVY CHAIN
2: A NOVEL INHIBITOR OF GLIOMA CELL INVASION IN
THREE-DIMENSIONS
T.E. Werbowetski, N.Y.R. Agar, and R.F. Del Maestro; Neurology and
Neurosurgery, Montreal Neurological Institute, Montreal, Canada
The highly infiltrative nature of malignant glioma makes surgical
removal difcult. There are many positive regulators of malignant brain
tumor growth and motility; however, little is known about inhibitory mod-
ulators of malignant glial cell invasion. Previous studies in our laboratory
suggest the presence of repellents and/or inhibitors in the serum-containing
conditioned medium from glioma cell lines (Werbowetski et al., J. Neuro-
biol. 60, 71, 2004). We have, therefore, developed and optimized a func-
tional screening assay using protein purication to isolate potential inhibi-
tors or repellents of glioma tumor invasion in three-dimensional collagen
gels from both endogenous and serum-derived sources. Serum-containing
conditioned medium from C6 rat astrocytoma spheroids from spinner cul-
ture was concentrated and applied to a Resource Q anion exchange column
on an AKTA FPLC. Fractions were collected and applied to C6 spheroids
implanted in a collagen matrix, and those that inhibited invasion were
pooled and applied to a heparin sepharose afnity column. Fractions were
again collected and subjected to the spheroid functional screening assay,
and the most inhibitory fractions were sent for mass spectrometry. Mass
spectrometry analysis of inhibitory fractions identied inter alpha trypsin
inhibitor heavy chain-2 (ITI-H2). This protein is produced by the liver,
secreted into serum, and acts alone or bound to the light-chain bikunin to
stabilize the extracellular matrix and/or inhibit serine protease activity in
the cumulus oocyte complex and in a variety of tumor cells in hyaluronic
acidrich environments to negatively regulate cell motility. An antibody
raised against ITI-H2 and tested using Western blot analysis suggests that
ITI-H2 is present as both a single protein and a bikunin-bound form in the
inhibitory fractions. Stable cell lines of ITI-heavy chain and bikunin overex-
pressing glioma and HEK-293 cell lines are currently being tested to further
validate our model both in vitro and in vivo. Our studies identify a role
for inter alpha trypsin inhibitor in malignant glial cell invasion and war-
rant further study of serum proteins as readily available sources of invasion
inhibitors. The identication of serum-derived inhibitors of invasion may
have potential therapeutic value in a variety of inltrative and metastatic
tumors in addition to malignant glioma.
295. BACITRACIN INHIBITS GLIOMA CELL MIGRATION
AND INVASION IN VITRO
D. Goplen,
1
J. Wang,
1
B.B. Tysnes,
1
A.J.A. Terzis,
4
O.D. Laerum,
2

and R. Bjerkvig
1,3
;
1
Department of Biomedicine, University of Bergen,
Bergen, Norway;
2
The Gade Institute, University of Bergen, Bergen,
Norway;
3
NorLux Neuro-Oncology, Bergen, Norway; NorLux Neuro-
Oncology, Centre de Recherche Public Sant, Luxembourg
By serial transplantation of human glioblastoma biopsies in nude rats,
two different tumor phenotypes are obtained, one that is highly invasive and
one that is predominantly angiogenic. Using a global proteomics approach
on the phenotypes, we identied a number of unique proteins that were dif-
ferentially expressed in the invasive phenotype. The proteins were identied
by peptide mass ngerprinting and bioinformatics and veried by Western
blots and on immunostained biopsy specimens. Protein disulde isomerase
(PDI) A6 precursor was identied as one of the overexpressed proteins in
the invasive phenotype. PDI is a chaperone protein that mediates integrin-
dependent cell adhesion. It is present both in the cytosol and at the cell
surface. Immunouorescent staining of glioma spheroids in vitro showed
that migrating cells expressed PDI. Since cell migration is a part of the
tumor invasion process and PDI was stronger expressed in invasive glioma
phenotype, we tested the effect of the PDI inhibitor bacitracin on migration
of glioma cells in different invasion assays. Both tumor spheroids derived
from human glioblastoma xenografts in the nude rat brain as well as cell
line spheroids were used. The human glioma cell lines (U373, U251, D54,
GaMg, and U87) expressed PDI. Bacitracin at concentrations as low as 5
mM inhibited tumor cell migration and invasion. The anti-invasive effect of
bacitracin was reversible after withdrawal of the inhibitor, indicating a spe-
cic, non toxic effect. In conclusion, using a global proteomics approach,
PDI was identied to play an important role in glioma cell invasion, and its
action was effectively inhibited by bacitracin.
296. SLIT2-ROUNDABOUT 1(ROBO1) SIGNALING INHIBITS
MEDULLOBLASTOMA BUT NOT GLIOMA CELL INVASION
IN THREE-DIMENSIONS
T.E. Werbowetski,
1
M. Seyed Sadr,
1
N. Jabado,
3
A. Angers-Loustau,
1

R. Bjerkvig,
4
J. Antel,
2
D. Faury,
3
Y. Rao,
5
and R.F. Del Maestro
1
;
1
Neurology and Neurosurgery and
2
Neuroimmunology Unit, Montreal
Neurological Institute, Montreal, Canada;
3
Department of Pediatrics,
Montreal Childrens Hospital Research Institute, Montreal, Canada;
4
Department of Anatomy and Cell Biology, University of Bergen, Bergen,
Norway;
5
Department of Neurology, Feinberg School of Medicine,
Northwestern University, Chicago, Illinois, USA
Chemotropic cues such as the Slit, Netrin and Semaphorin families
guide the migration of neuronal and glial cell precursors during neural
development. Recently, Slit and its receptor Roundabout (Robo) have been
implicated in tumor angiogenesis and leukocyte migration. It is not known
if these molecules contribute to directing the invasion of brain tissue by
medulloblastoma and glioma cells. The biology of brain neoplasms such
as medulloblastoma and malignant glioma has provided a link between
tumorigenesis and neurodevelopment. Recent studies placing brain tumors
in the context of neurodevelopment have led to the recognition of new
tumor suppressors and oncogenes involved in tumor progression. The cur-
rent study focuses on the expression and functional signicance of Slit2
and Robo1 in medulloblastoma and glioma tumors. Invasion assays were
carried by out using time-delayed co-culture, sodium alginate bead micro-
encapsulation, time-lapse videomicroscopy, and confrontation co-culture
with astrocyte aggregates in collagen type I matrices. RT-PCR was used
to examine Slit2 and Robo1 expression levels in glioma and medulloblas-
toma cell lines and primary human tumors. Here, we provide evidence
that medulloblastoma cells are inhibited, but not repelled, by a localized
concentration of Slit2 in collagen gels as demonstrated by time-delayed co-
culture, sodium alginate bead microencapsulation, and confrontation co-
culture with astrocyte aggregates. Slit2 had no signicant effect on glioma
cell invasion in all models tested. Medulloblastoma and glioma cell lines
express Slit2 and its receptor Robo1, and both functional blocking antibody
and dominant negative Robo experiments demonstrate a 50% rescue of
the invasive phenotype compared with Slit treatment alone. In addition,
Slit2 had an inhibitory effect on medulloblastoma cell proliferation, but
did not affect glioma cell doubling time. Studies are currently underway
to assess the effect of RNAi knockdown of Robo1 in medulloblastoma cell
lines with the goal of rescuing the inhibitory effect of Slit2. The signal
transduction machinery downstream of Slit-Robo is also currently being
dissected in medulloblastoma and glioma cell lines. Our ndings indicate
that the effect of Slit2 is not conserved for all types of brain tumors, and
that manipulation of Slit-Robo signaling may serve as a potential treatment
for medulloblastoma tumors.
297. EGFR-MEDIATED ACTIVATION OF PHOSPHOLIPASE
C GAMMA IS ASSOCIATED WITH INCREASED
GLIOBLASTOMA MOTILITY/INVASION
J. Uhm,
1
K. Aldape,
2
A. Yates,
3
J. Goble,
1
A. Nilson,
1
L. Frederick,
1
and
C.D. James
1
;
1
Neurology, Mayo Clinic Rochester, Rochester, Minnesota;
2
M.D. Anderson Cancer Center, Houston, Texas; USA
EGFR alterations represent one of the most frequent gene alterations
in glioblastoma. We have previously shown that EGFR activation in vitro
stimulates glioma motility/invasion by way of increased PLC-gamma acti-
vation. Conversely, inhibition of PLC gamma by pharmacologic means
(U73122) or by small interfering RNA (siRNA) abrogrates EGFR-induced
motility. We further evaluated the relationship between EGFR and PLC
phosphorylation in vivo at two levels. First, in both orthotopic (intracra-
nial) and heterotopic (subcutaneous ank) xenografts, EGFR-amplied
xenograft tumors demonstrated PLC-gamma activation, in contrast to
tumors not amplied for EGFR. We also evaluated 89 human glioblastoma
specimens and found a very signicant correlation between EGFR ampli-
cation and PLC gamma phosphorylation: 16/31 EGFR amplied tumors
versus 6/58 EGFR nonamplied tumors (P 0.0001). Of the EGFR ampli-
ed tumors, those with amplication of EGFRvIII (deleted for codons 6
273) showed a higher association with PLC gamma phosphorylation (8/10)
than tumors with wild-type EGFR amplication (8/21). Taken together,
our results support that a relationship exists between high-level, amplica-
tion-associated expression of EGF receptor and PLC gamma activation and
that PLC-gamma activity may be an important mediator of glioblastoma
cell invasion. Hence, EGFR (especially the vIII form) as well as PLC gamma
may represent viable targets for anti-invasive therapeutics.
298. REGULATION OF GLIOMA INVASION BY A PAIR
OF INTERACTING AND FUNCTIONALLY OPPOSING
PROTEINS IGFBP2 AND IIP45
W. Zhang, G. Fuller, S. Song, and G. Wang; Pathology, M.D. Anderson
Cancer Center, Houston, Texas, USA
Cancer cell invasion, often a rst step in cancer metastasis, is a major
factor contributing to the morbidity and mortality of cancer. In diffuse glio-
mas, the high mortality rate is primarily a result of local invasion. Thus, a
detailed understanding of how tumor invasion is regulated is critical. Using
genomic and proteomic approaches, we discovered that insulin-like growth
factor binding protein 2 (IGFBP2) is overexpressed in 80% of glioblastoma.
Cell biology studies and animal model experiments showed that IGFBP2
enhances glioma invasion through activation of integrin signal transduction
pathways. Using yeast two-hybrid system, we discovered a new protein that
binds to IGFBP2. Our in vitro assays and xenograft mouse model experi-
ments functionally characterized this novel gene as an inhibitor of glioma
invasion (Song et al., Proc. Natl. Acad. Sci. USA 100, 13970, 2003). We
named this gene IIp45, which stands for Invasion Inhibitory protein with a
molecular mass of 45 kDa. In recent studies, we found that the IIp45 gene
is alternatively spliced in a tumor-specic manner. The alternatively spliced
transcript produces an isoform, IIp45S, which has a distinct C-terminal
region due to a frame-shift. The tumor specic isoform IIp45S is unstable
and is rapidly degraded by the uniquitin-proteasome degradation pathway.
Thus, we have revealed two interacting and functionally opposing proteins,
IGFBP2 and IIp45, which play an important role in regulation of invasion.
Elevation of IGFBP2 and inactivation of IIp45 are two key events that con-
tribute to the highly invasive phenotype of glioblastoma.
299. CELL INVASION OF HUMAN PITUITARY ADENOMA
CELL LINE, HP-75 IN HYPOXIA
D. Yoshida, K. Kim, M. Noha, and A. Teramoto; Neurosurgery, Nippon
Medical School, Tokyo, Japan
Pituitary adenoma tissue is known as hypovascular, and concomitantly
the partial oxygen pressure is lower than the surrounding normal organs
likely in the other cancer tissues. In this study, we investigated whether
hypoxia inuences the cell invasiveness of the human nonfunctioning pitu-
itary adenoma cell line, HP-75. HP-75cells were exposed to hypoxia (1%
for 24 h). The subsequent mRNA expression of genes was examined by
cDNA microarray. The results were veried by real-time RT-PCR. Gela-
tin zymogram and reverse zymogram were employed to determine enzyme
activities of MMP and TIMP. Cell motility, chemotaxis, and haptoinva-
sion were studied with Boiden chamber. Cell adhesion assay and cell-to-cell
adhesion were further studied. Cyclic DNA microarray and real-time RT-
PCR indicated that laminin -2 chain mRNA was specically upregulated
by hypoxia (4.16-fold), but not in the other genes relating to cell motility
and invasion involving extracellular matrix, cell adhesion molecules, and
MMP families. Immunouorescent study also demonstrated the increased
expression of laminin -2 chain at the protein level followed by hypoxic
induction. Gelatin zymogram and reverse zymogram, showing MMP and
TIMP activities, were not particularly changed. Haptoinvasion and chamo-
taxis or cell adhesion to collagen type IV were elevated (12.8-, 6.8-, or
8.8-fold). Meanwhile, cell adhesion and chemotaxis to laminin, collagen
type I, and bronectin were not modulated by hypoxia. Cell motility was
not amended by hypoxia. Cell-to-cell adhesion was signicantly elevated
(9.6-fold). These results highly suggest that hypoxia induces elevated cell
invasion and cell to cell mediated by elevated transcription of laminin -2
molecule that is specically bound to collagen type IV.
300. HEMANGIOGENIC PHENOTYPES AS SURROGATE
BIOMARKERS IN BRAIN TUMOR TREATMENT
P. Gutin,
1
D. Jin,
2
P. Lau,
2
L. Vincent,
2
K. Shido,
2
P. Salerno,
1
and S.
Rai
2
;
1
Neurosurgery Service, Memorial Sloan-Kettering Cancer Center;
2
Department of Genetic Medicine, Weill Medical College of Cornell
University, New York, New York; USA
No biomarkers are currently available to indicate the angiogenic pro-
pensity of brain tumors nor to evaluate their response to therapy. Nev-
ertheless, many different antiangiogenic drugs are currently being evalu-
ated in clinical trials. Here we describe a novel in vitro functional assay,
HUVEC-based angiogenic scale (HBAS), to assess the overall angiogenic
activity in the plasma of patients with glioma and meningioma. Analysis
of a cohort of 50 consecutive patients with these tumors showed that the
majority had higher HBAS at baseline prior to surgical resections than age-
matched normal subjects. The plasma HBAS also correlated with increased
plasma and cellular vascular endothelial growth factor-A (VEGF-A) levels
by ELISA. The cell lysate fraction from platelets was found to have the
highest level of pro-angiogenic activity. In addition, comparison of heman-
giogenic cellular markers indicated a switch toward increased circulating
CD133
VEGFR2
endothelial progenitor cells (by ow cytometry) and

circulating hematopoietic stem/progenitor cells (by methylcellulose colony
forming assays) in subsets of patients with active glioma or meningioma.
This hemangiogenic switch also correlated with an increased plasma level
of stromal-derived factor-1 alpha (SDF-1a), suggesting that mobilization of
these hemangiogenic progenitors from the bone marrow to the brain tumor
neo-angiogenic niche may be at least partially mediated by the chemoki-
netic SDF-1a/CXCR4 pathway. Work is underway to study the correlation
between the hemangiogenic phenotypes and the clinical outcome of these
patients. Taken together, collective assessment of hemangiogenic biomark-
ers is a promising tool to provide valid end points for future clinical trials
for patients with glioma and meningioma.
301. VEGF DOES NOT PROTECT ENDOTHELIAL CELLS, OR
GBM- CELLS, AGAINST IRRADIATION
M. Donker,
1
W. van Furth,
2
K. Hovinga,
2
C. van Bree,
1
D. Bosch,
2
and
L. Stalpers
1
;
1
Radiation Therapy;
2
Neurosurgery, Academic Medical
Centre, Amsterdam, The Netherlands
In a number of recent publications, VEGF has been implicated not only
as a stimulator of angiogenesis, but also as a survival factor for both endo-
thelial cells and GBM cells after irradiation. GBM is a highly resistant tumor
with a high VEGF secretion. We hypothesize that VEGF protects endothe-
lial cells and GBM cells against damage of ionizing radiation. Four different
types of endothelial cells were used, commercially available HUVECs (from
Clonetics), a HUVEC cell line (EC-RF24), primary HUVECs and bovine
retina endothelial cells (BRC), and two GBM cell lines (Gli-06 and U87).
Cells were irradiated with 0, 2, or 5 Gy under low-serum conditions with
three different concentrations of VEGF added to the medium. Cell survival
after single dose irradiation was measured by the XTT proliferation assay
after 96 h. In addition, four human GBM cell lines (U87, Gli-06, U251,
and U251-NG2) were irradiated with various single dose fractions between
0 Gy and 20 Gy. The VEGF concentration in the medium was measured
by ELISA at 0, 24, 48 and 72 h after gamma-irradiation. All cells tested
showed already inhibited cell proliferation after single dose irradiation of
2 Gy. Although VEGF stimulated endothelial cell proliferation in a dose
dependent manner, the cell-killing effect of irradiation was not affected.
The variation in radiosensitivity of endothelial cells was smaller than that of
GBM-cells, with U87 being the most radioresistant. Interestingly, in all GBM
cell lines we found a dose-dependent increase in the VEGF-secretion after
ionizing radiation, with U87 having a 8-times higher VEGF secretion than
the other tested cell lines, with a twofold increase in VEGF secretion 72 h
after a single dose of 20 Gy. We could not demonstrate a protective effect
of VEGF on cell death after ionizing irradiation of endothelial cells, nor
GBM cells. We used four different types of endothelial cells to model brain
tumor endothelial cells. The endothelial cells showed proliferative effects
of VEGF, suggesting an active VEGF-R1 system. The discrepancy with the
literature will be discussed. We conclude that VEGF is not a survival factor
for endothelial or GBM cells.
302. ANTITUMOR EFFECTS OF A COMBINATION
THERAPY CONSISTING OF IRRADIATION AND COX-II
INHIBITION IN AN ORTHOTOPIC, SYNGENEIC MURINE
GLIOMA MODEL
M. Wagemakers,
1
G. van der Wal,
2
J. Mooij,
1
and G. Molema
2
;
1
Neurosurgery, Groningen University Hospital, Groningen;
2
Pathology
and Laboratory Medicine, Medical Biology section, Groningen
University Institute for Drug Exploration and University Hospital
Groningen, Groningen; The Netherlands
COX-II inhibitors and irradiation have been demonstrated to act syner-
gistically in rodent tumor models, among others in a subcutaneous glioma
model. Controversy exists regarding the molecular basis for this phenom-
enon and whether the tumor cells, tumor vasculature, host immune cells,
or a combination of these is primarily affected. We initiated a study to
investigate the presence of synergistic effects of COX-II inhibition and ion-
izing radiation treatment of intracranial glioma, and to analyze the possible
role of tumor endothelial cells in this process. Syngeneic, COX-II negative,
GL261 tumor cells were injected intracranially into C57bl6 mice. Admin-
istration of a COX-II inhibitor or vehicle control was started 15 days after
tumor cell inoculation. Local fractionated irradiation (5 3 Gy) or sham
procedures were performed starting at day 22. Animals were terminated
4 weeks after inoculation of tumor cells. Tumor volume was determined,
and cryostat cut sections were immunohistochemically stained for endo-
thelial cells activation markers, and leucocytes. Tumor and brain tissues
were furthermore prepared for quantitative RT-PCR analysis regarding
mRNA expression levels of VEGF, VEGF receptor, angiopoietin-1 and -2,
Tie-2, and av integrin. Preliminary results show a trend toward a radiosen-
sitizing effect of COX-II inhibitor treatment. COX-II inhibition alone was
devoid of an antitumor effect. All tumors intensively expressed ICAM-I.
Irradiation affected leucocyte inltration into the tumors. Analysis of the
mRNA expression levels of the above-mentioned genes is ongoing. There is
a trend toward synergy between COX-II inhibition and irradiation in the
treatment of intracranial glioma in this model. Experiments are ongoing
to further validate these rst observations of a radiosensitizing effect of
COX-II inhibitors, and to analyze the molecular basis of this effect in in
vivo and in vitro studies.
303. SYNERGISTIC EFFECTS OF THE VEGF-R TYROSINE
KINASE INHIBITOR ZD6474 AND RADIOTHERAPY ON
TUMOR GROWTH IN THE INTRACEREBRAL BT4C RAT
GLIOMA MODEL
M. Sandstrm,
1
P. Bergstrm,
1
M. Johansson,
1
and R. Henriksson
1,2
;
1
University Hospital Ume, Ume;
2
AstraZeneca AB; Sweden
Malignant glioma is characterized by extensive pathological neovas-
cularization. Vascular endothelial growth factor (VEGF) is commonly
believed to be the key positive regulator of glioma angiogenesis. ZD6474 is
a potent, orally active, low-molecular-weight inhibitor of VEGF receptor
tyrosine kinase activity with additional inhibitory effects on the epidermal
growth factor (EGF) receptor tyrosine kinase. ZD6474 has previously been
reported to inhibit tumor growth and neovascularization in a broad panel
of tumor xenografts. In a previous study we show that ZD6474 signicantly
inhibits tumor growth in an orthotopic intracerebral glioma model. In the
present study we have investigated if ZD6474 in combination with radio-
therapy has any synergistic effects on tumor growth in an intracerebral rat
glioma model. The effects of ZD6474 and radiotherapy upon tumor growth
were investigated in the intracerebral BT4C rat glioma model. Animals were
randomized into four groups, with 7 animals in each group. One group
was untreated. A second group received ZD6474 30 mg/kg daily as an oral
gavage for 14 days, starting day 6 after tumor implantation. A third group
received 12 Gy single fraction radiotherapy at day 6. Finally, the fourth
group was treated with ZD6474 30 mg/kg for 14 days and radiotherapy
12 Gy at day 6, in combination. Animals were sacriced on day 20 and
tumor size was measured. ZD6474 30 mg/kg in combination with radio-
therapy signicantly decreased tumor area from 16 mm
2
(range, 428) to
6 mm
2
(range,

210) (P 0.05) when compared to untreated controls. The
orally available VEGFR2 receptor tyrosine kinase inhibitor ZD6474 inhib-
its tumor growth in an intracerebral rat glioma model. Combination with
radiotherapy results in more than additive effects. These results reported
justify further experimental investigations on the effects of ZD6474 in
malignant glioma.
304. ASSESSING U251 CLONAL POPULATIONS AT THE
CELLULAR AND MOLECULAR LEVELS
D. Maret and R.F. Del Maestro; Neurology and Neurosurgery, Montreal
Neurological Institute, Montreal, Canada
Malignant gliomas are highly invasive tumors that exhibit heterogene-
ity of morphology and behavior. We have isolated clonal populations of
the U251 cell line and analyzed these clones at the cellular and molecular
levels. Clonal populations of wild-type U251 were isolated and expanded.
Spheroids composed of individual clones were implanted into a type I col-
lagen gel to assess invasion in three dimensions. Time-lapse microscopy was
used to monitor the directional invasion and mitosis of individual cells. The
mitotic activity observed was compared with Ki-67 staining and doubling
times of monolayer cultures. Clonal populations exhibiting differences in
invasion are being studied at the molecular level, by differential protein
expression proling. We have determined that the U251 clones fall into
three categories of invasiveness: hypo-, hyper-, and intermediate invasion.
We found that the U251 clones do not defer cell proliferation for invasion
in a type I collagen matrix. Individual cells were found to invade a clonal-
dependent distance regardless of the number of divisions taking place. This
is contrary to the Go or Grow hypothesis, which proposes that cell divi-
sion and cell migration are temporally exclusive events and that tumor cells
defer cell division to migrate. Plasma membrane proteins have been isolated
from hyper- and hypo- invasive clones, and the enrichment and purity have
been assessed. Multiple clonal populations are present in glioma cell lines
harboring distinct invasive and mitotic characteristics. Invasion studies car-
ried out by using glioma cell lines are the combined results of heterogeneous
populations and do not accurately reect the individual clonal populations
present. Our differential protein expression analysis will identify key pro-
teins involved in invasion, which have the potential to serve as therapeutic
targets.
305. MAP2K3 AND P38 ARE DRIVERS OF IN VITRO GLIOMA
INVASION
T. Demuth,
1
J. Rennert,
1
D. Hoelzinger,
1
R. Reavie,
1
L. Mariani,
2
and
M. Berens
1
;
1
Brain Tumor Unit, Neurogenomics Department, TGen,
Phoenix, Arizona, USA;
2
Switzerland
The early and pervasive tendency of glioma cells to invade into peri-
tumoral normal brain underlies the poor prognosis for patients with glial
tumors. This malignant dispersion of glioma prevents complete surgical
resection, positions tumor cells behind an intact blood-brain barrier, and
sequesters tumor cells outside the elds of focal radiation; each of these
leads to heightened (almost certain) tumor recurrence. A three-dimensional
spheroid invasion assay was employed to determine the transcriptome of
invasive glioma cells (U87WT and U87EGFR) compared to their non-
invading counterparts from the spheroid center. Cells from invasive rim
and spheroid core were collected by laser capture microdissection as three
biological replicates representing each cell line; mRNA was isolated and
underwent oligonucleotide microarray analysis. Mitogen-activated protein
kinase kinase 3 (MAP2K3), a member of the MAP-kinase family, was iden-
tied to be signicantly upregulated in invasive cells. MAP2K3 is involved
in stress signaling, tumor cell invasion, and apoptosis resistance; MAP2K3
activates p38 by phosphorylation. Immunouoresence on sections from par-
afn-embedded spheroids in the 3D invasion assay revealed increased levels
of MAP2K3 and phosphorylated p38 in invasive cells. Inhibition of these
genes by siRNA and small molecules decreased invasiveness of spheroids in
vitro while sensitizing them to apoptosis induction, conrming signicance
of this pathway for glioma invasion. Glioma invasion tissue microarray
staining revealed strong intensity for MAP2K3 and phosphorylated p38 in
100% of invasive glioma cells, while cells from the core exhibited weak or
no staining; noncancerous brain revealed no staining for MAP2K3. This
data suggests that MAP2K3 and p38 are potential targets for anti-invasive
therapies in combination with cytotoxic agents.
306. MICROARRAY-BASED COMPARATIVE GENOMIC
HYBRIDIZATION ANALYSIS OF ASTROCYTIC TUMORS
OCCURRING IN A LI-FRAUMENI-LIKE SYNDROME FAMILY
R.M. Reis,
1
B. Carvalho,
2
J. Amorim,
3
S.A. Ribeiro,
1
F. Pardal,
4
R.
Almeida,
5
and B. Ylstra
6
;
1
Life and Health Sciences Research Institute
(ICVS), Health Sciences School, University of Minho, Braga, Portugal;
2
Department of Pathology, Free University Medical Center, Amsterdam,
The Netherlands; Departments of
3
Internal Medicine,
4
Pathology, and
5
Neurosurgery, Hospital S. Marcos, Braga, Portugal;
6
Microarray Core
Facility, Free University Medical Center, Amsterdam, The Netherlands
Li-Fraumeni syndrome (LFS) is a rare autosomal dominant disease. It
is characterized by a familiar clustering of a wide range of cancers, pre-
dominantly breast cancer, sarcomas, brain tumors, and adrenal cortex
cancer, diagnosed before the age of 45 years. Two distinct forms of LFS
can be recognized, a classic LFS and a Li-Fraumeni-like syndrome (LFL).
In the majority of the LFS families, the underlying genetic defect is a germ
line mutation in TP53 gene. Here, we describe a LFL family characterized
by the presence of brain tumors in two siblings, a 26-year-old man with
three metachronous astrocytic tumors (two diffuse astrocytomas and one
glioblastoma, which occurred within a 4-year interval) and his 30-year-old
sister with one diffuse astrocytoma. The advent of microarray-based com-
parative genomic hybridization (array-CGH) technology allows the analysis
of whole chromosomal aberrations in a single experiment with high spatial
resolution. The aim of this study was to assess the presence of TP53 germ
line mutations in this LFL family and to relate it with the chromosomal
aberrations of the astrocytic tumors. TP53 germ line mutation analysis of
exons 58 was performed by PCR followed by direct sequencing of DNA
isolated from blood of affected siblings. From the formalin-xed, parafn-
embedded histological sections of the four astrocytic tumors, DNA was
isolated and used for microarray-CGH analysis. The array-CGH consisted
of about 5000 BAC clones with an average resolution of 1 Mb. Identical
germ line TP53 exon 8, G871A, Arg290His mutation was identied in both
siblings. The microarray-CGH analysis of the three metachronous tumors
of the male has as follows: The rst diffuse astrocytoma did not show clear
chromosomal alterations; the second diffuse astrocytoma showed some
chromosomal alterations, including losses on chromosome 6q, 10q, and 13
and gain on 5q region; the glioblastoma showed the aberrations detected in
the previous astrocytoma and contained additional abnormalities, includ-
ing losses on 2p, 3p, 5p, 10q, 11p, 13p, 14q, 16q, and 22, as well as gain on
5p and 10p. The microarray-CGH analysis of the diffuse astrocytoma of
the female showed no clear chromosomal aberrations. This study reports a
TP53 germ line mutation in an LFL family associated with brain tumors in
young adults. Microarray-CGH analysis of tumors shows the presence of
chromosomal alterations, such as loss on 2p, 3p, and 16q and gain on 5q
regions that are not usually present in sporadic astrocytic tumors.
307. CHANGES IN PROTEIN EXPRESSION FOLLOWING
RADIOTHERAPY IN EXPERIMENTAL MALIGNANT
GLIOMA
C. Wibom,
1
F. Pettersson,
2
M. Johansson,
1
R. Henriksson,
1
and A.T.
Bergenheim
3
;
1
Oncology;
2
Organic Chemistry;
3
Neurosurgery, University
Hospital Ume, Ume, Sweden
The outcome of modern treatment for glioblastoma (GBM) has so far
been disappointing. In order to develop new GBM treatment modalities,
as well as to improve present ones, a more detailed understanding of the
biological effects of different treatments must be acquired, together with
new tools for a more rapid assessment of these effects. Today, radiotherapy
is one of the mainstays of GBM treatment. This study aims to characterize
differences in protein expression patterns in brain tumor tissue following
radiotherapy in an experimental rat glioma model. BT4C-cells were stereo-
tactically implanted into the right nucleus caudatus of 24 BD IX-rats. One
group received radiotherapy delivered as a 12-Gy single fraction on day 12
after tumor implantation. On day 1, 5, 7, and 12 after irradiation, three
animals from each group were sacriced, and tumor tissue from each ani-
mal was analyzed with regard to protein expression using surface-enhanced
laser desorption/ionizationtime of ightmass spectrometry (SELDI-TOF-
MS). Mass spectrometric data was analyzed with principle components
analysis (PCA) to detect differences between the groups as well as possible
temporal changes. Using PCA, regions of interest within mass spectrograms
of 2.550 kDa were identied and further characterized through compari-
sons of mean peak intensities between the groups. Univariate F-test sta-
tistics revealed several peaks whose intensity signicantly changed after
radiotherapy. The prompt changes in the protein expression are a novel
observation and might be of value to understand biological events follow-
ing irradiation. The SELDI-TOF-MS technique in conjunction with PCA
seems to be a well suited tool to study these changes and can detect specic
proteins displaying differentiated expression levels. In a further perspec-
tive these ndings may prove to be useful in the development of new GBM
treatment schedules.
308. MGMT METHYLATION STATUS AND EXPRESSION
LEVEL DO NOT CORRELATE WITH SENSITIVITY TO CCNU
IN SHORT-TERM CULTURES DERIVED FROM MALIGNANT
ASTROCYTOMA
T. Warr,
1
R. Poh,
1
B. Suarez-Merino,
1
S. Ward,
1
P. Warren,
3
J. Darling,
3

and D. Thomas
2
;
1
Department of Molecular Neuroscience and
2
Division
of Neurosurgery, Institute of Neurology, University College London,
London;
3
University of Wolverhampton, Research Institute of Healthcare
Science, Wolverhampton; UK
Adjuvant chemotherapy using DNA-damaging agents has largely failed
to make a signicant impact on the outcome of patients with malignant
astrocytoma. One of the primary mechanisms of resistance to nitrosureas
such as CCNU is mediated through O
6
-methylguanine-DNA methyltrans-
ferase (MGMT). This DNA repair enzyme removes the cytotoxic alkyl
adducts from O
6
-guanine, and hence the level of MGMT activity in tumor
cells is related to their sensitivity to nitroureas. It has been proposed that
functional inactivation of MGMT through hypermethylation of the gene
promotor region could be predictive of chemosensitivity. We have previ-
ously reported differential sensitivity to CCNU in a panel of 17 short-term
cultures derived from malignant astrocytoma. In this study, we determined
the methylation status of MGMT using methylation-specic PCR in these
17 cultures. We also assessed the amounts of MGMT mRNA and protein
present in each culture using real-time quantitative PCR and immunohis-
tochemistry with a commercial antibody against MGMT. There was good
correlation between MGMT promotor methylation and presence of MGMT
mRNA and protein in all but 2 cases. In both these cultures, mRNA and
protein were not detected even though the MGMT promotor was unmeth-
ylated. However, there was no correlation between sensitivity to CCNU
and MGMT status. In the 2 most resistant cultures, the MGMT gene was
methylated and was not expressed. Similarly, in 4/5 of the most sensitive
cultures, MGMT was unmethylated, and in 2 of these cases, there was
commensurate MGMT expression. However, in the remaining 2 cultures,
MGMT expression was not detected, indicating that an alternative mecha-
nism to gene methylation is responsible for MGMT inactivation. This study
highlights that the resistance of malignant astrocytoma to nitroureas may
be more complex than simple reliance on MGMT activity and prediction
of response to such agents by MGMT methylation status should be used
with caution.
309. SHARED EPIGENETIC MECHANISMS OF GENE
INACTIVATION IN HUMAN AND MOUSE GLIOMAS
J.F. Costello,
1
C. Hong,
1
P. Jun,
1
A. Maunakea,
1
W.A. Weiss,
3
and A.W.
Bollen
2
;
1
2
Pathology, and
3
Neurology, University
of California San Francisco, San Francisco, California, USA
Human tumors arise from the deleterious effects of genetic and epige-
netic mechanisms on gene expression. In several mouse models of human
tumors, the tumorigenic phenotype is reversible, suggesting that epige netic
mechanisms also contribute signicantly to tumorigenesis in mice. It is
not known whether these are the same epigenetic mechanisms in human
and mouse tumors, or whether they affect homologous genes. Using an
integrated approach for genome-wide methylation (epigenetic) and copy
number (genetic) analyses, we identied SLC5A8 on chromosome 12q23.1
and PRKWNK2 on chromosome 9q22.31 that were affected primarily by
aberrant methylation in human astrocytomas and oligodendrogliomas.
SLC5A8 encodes a sodium/monocarboxylate cotransporter, and PRK-
WNK2 encodes a putative serine/threonine kinase. Both genes are highly
expressed in normal brain but significantly downregulated in primary
gliomas. Bisulte sequencing analysis showed that their promoter CpG
islands were unmethylated in normal brain, but extensively methylated in
brain tumors, consistent with the tumor-specic loss of gene expression. In
glioma cell lines SLC5A8 and PRKWNK2 expression was also suppressed
but could be reactivated with a methylation inhibitor. Expression of exog-
enous SLC5A8 or PRKWNK2 in glioma cells inhibited colony formation,
suggesting they may function as growth suppressors in vitro. Remarkably,
9 of 10 murine oligodendroglial tumors (from p53+/- or ink4a/arf+/- ani-
mals transgenic for S100b-v-erbB) demonstrated a similar tumor-specic
downregulation of mSCL5A8, the highly conserved mouse homologue
of SLC5A8. Similarly, the murine PRKWNK2 was also methylated and
downregulated in a proportion of the mouse gliomas. Taken together, these
data suggest that SLC5A8 and PRKWNK2 function as growth suppres-
sors in vitro and that epigenetic mechanisms are their primary cause of
gene silencing in human gliomas. The shared epigenetic inactivation of both
SCL5A8 and PRKWNK2 in mouse and human gliomas indicates an addi-
tional degree of commonality in the origin and/or pathway to tumorigenesis
between primary human tumors and these mouse models of gliomas.
310. GENETIC ALTERATIONS IN DESMOPLASTIC
MEDULLOBLASTOMAS: EVIDENCE FOR MONOCLONAL
TUMOR ORIGIN AND IDENTIFICATION OF NOVEL
AMPLIFIED AND OVEREXPRESSED PROTO- ONCOGENES
A. Ehrbrecht,
1
U. Mueller,
2
M. Wolter,
3
A. Hoischen,
1
B. Radlwimmer,
2

T. Pietsch,
4
P. Lichter,
2
G. Reifenberger,
3
and R. Weber
1
;
1
Institute
of Human Genetics, Bonn;
2
German Cancer Research Center,
Rheinische Friedrich-Wilhelms-University, Heidelberg;
3
Department
of Neuropathology, Heinrich Heine University, Duesseldorf;
4
Institute
of Neuropathology, Rheinische Friedrich-Wilhelms-University, Bonn;
Germany
Desmoplastic medulloblastomas (dMBs) are histologically character-
ized by two distinct tumor components, the so-called pale islands and the
desmoplastic areas. Previous molecular studies have shown that dMBs
frequently carry PTCH mutations. However, little is known about other
genetic and chromosomal aberrations associated with these tumors. We
investigated total tumor DNA of 23 sporadic dMBs using comparative
genomic hybridization (CGH). Chromosomal imbalances were identied
in 17 tumors (74%). The number of aberrations detected per tumor varied
from 1 to 12, with an average of 4.61 0.73 (mean SEM). Recurrent
chromosomal gains were detected on chromosomes 3 and 9 (6/23); 2 and
20 (5/23); 6, 7, 17, and 22 (4/23 each); and 1 (3/23). Recurrent losses were
found on chromosomes X (8/23); Y (6/13 male patients); 9 and 12 (4/23
each); as well as 10, 13, and 17 (3/23 each). Amplications were detected in 4
tumors and mapped to 1p22, 5p15, 9p, 12p13, 13q33-q34, and 17q22-q24.
To address the question of clonality of the two components in dMBs, we
performed CGH analysis on microdissected pale islands and desmoplas-
tic areas. In 5/6 informative tumors both histological components shared
common chromosomal imbalances, indicating an origin from a single pro-
genitor cell. We additionally characterized the amplicons detected on 5p15,
9p, and 17q22-q24 in 2 dMBs using matrix-CGH on genomic arrays of
6.000 large insert clones. Subsequent molecular analyses of amplied can-
didate genes identied by matrix-CGH conrmed amplication of several
genes on 17q23 in three dMBs and the JMJD2C gene on 9p24 in one dMB,
respectively. Expression analysis suggested RPS6KB1 as the most impor-
tant target on 17q23, which was found to be markedly overexpressed in
10/11 medulloblastomas investigated. Taken together, our study provides
strong genetic evidence for a monoclonal origin of dMBs and implicates
RPS6KB1 and JMJD2C as novel proto-oncogenes that are aberrantly acti-
vated in these tumors.
311. AMPLIFICATIONS AND DELETIONS IN THE
GLIOBLASTOMA GENOME: FROM NOVEL LOCI TO
CANDIDATE GENE AND NON- GENE TARGETS
C. Brennan,
1
B. Feng,
2
D. Louis,
3
P.M. Black,
4
and L. Chin
2
;
1
Neurosurgery, Memorial Sloan-Kettering Cancer Center, New York,
New York;
2
Medical Oncology, Dana Farber Cancer Institute, Boston,
Massachusetts;
3
Pathology, Massachusetts General Hospital, Boston,
Massachusetts;
4
Neurosurgery, Brigham and Womens Hospital,
Boston, Massachusetts; USA
Array-based comparative genomic hybridization (ACGH) offers increas-
ing resolution of amplication and deletion events in the tumor genome. The
targets of less common events, along with the signicance of these events,
are the subject of intense investigation. We present the combined analysis
of ACGH and expression proling of 40 primary glioblastomas and 20 cell
lines in an effort to identify narrow, high-copy-number aberrations which
point to a limited number candidate gene and non-gene targets, such as
micro-RNAs. Proles were generated using cDNA microarrays (Agilent)
providing an interval resolution of approximately 100 kb. A changepoint
algorithm (circular binary segmentation) was used to identify discrete copy
number aberrations (CNAs) and their boundaries. Locus boundaries are
systematically dened by grouping CNAs across multiple proles. Within
each locus, one or more minimal common regions (MCRs) of overlapping
alteration are automatically identied, each potentially harboring a distinct
cancer-relevant target. Twenty cell lines and 14 tumors were additionally
proled for RNA expression (Affymetrix). Expression data were mapped to
genome position, and each gene was tested for copy-number-driven expres-
sion by calculating the shift in expression in samples with CNA. Signi-
cance was estimated by permutation testing. One hundred sixty-four dis-
crete autosomal loci were identied, 111 present in more than one sample.
Many MCRs were of low-level copy number alteration. Fifty-ve MCRs
met high-condence criteria: high-level alteration and/or high recurrence.
Average size of this subset was 3.5 Mb, spanning an average of 36 genes.
Fifteen of these MCRs were present only in cell lines; 40 were identied in
tumors as well (21 amplications, 19 deletions). Supporting the validity of
the approach, all common CNAs previously described in high-grade glio-
mas were identied, including amplications of PDGFRA, EGFR, MDM2,
and CDK4 and deletions of p16(Ink4a) and PTEN. Aside from these, the
majority of loci were novel, either not previously described in glioma or
without characterized target genes. Eighteen loci spanned 1 Mb, contain-
ing only 20 genes. Each of these smaller loci was subject to quantitative
PCR validation. We identied several candidate targets, including TERT,
not previously identied as a target of amplication. Genes within the 52
loci were evaluated for expression, and 520 out of 1740 were found to show
signicant effect of copy number on gene expression. Results are contrasted
with identical analysis of 300 samples of 4 other non-glioma tumor types.
High-grade gliomas harbor infrequent novel chromosomal copy aberrations
which mark potential cancer-relevant genes. Expression proling allows
further narrowing of this list of targets by characterizing the response to
gene dosage.
312. COMPREHENSIVE GENETIC CHARACTERIZATION OF
PLEOMORPHIC XANTHOASTROCYTOMAS
A. Hoischen,
1
M. Ehrler,
1
K. Kaulich,
3
B. Blaschke,
3
P. Zipper,
3
A.
Becker,
2
B. Radlwimmer,
4
O. Wiestler,
4
P. Lichter,
4
G. Reifenberger,
3

and R. Weber
1
;
1
Institute of Human Genetics and
2
Department
of Neuropathology, Rheinische Friedrich-Wilhelms-University,
Bonn;
3
Department of Neuropathology, Heinrich-Heine-University,
Duesseldorf;
4
Department of Molecular Genetics, German Cancer
Research Center, Heidelberg; Germany
Pleomorphic xanthoastrocytomas (PXAs) are rare astrocytic neoplasms
corresponding histologically to WHO grade II. They usually show circum-
scribed growth and favorable prognosis despite exhibiting a high degree
of cellular pleomorphism. PXAs mainly affect children and young adults.
Here we present genomic profiling experiments of 50 PXAs. Chromo-
somal-CGH revealed a distinct pattern of chromosomal imbalances. The
hallmark alteration detected in 50% of PXAs was loss on chromosome
9. Less common recurrent losses affected chromosomes 17 (10%); 8, 18
and 22 (4% each); and the Y chromosome in 7.7% of tumors from male
patients. Recurrent gains were identied on chromosome X (16%); 7, 9q,
and 20 (8% each); and 4, 5, and 19 (4% each). Amplications were found
in 2 tumors and mapped to 2p23-p25, 4p15, 12q13, 12q21, 21q21, and
21q22. To achieve a higher resolution, 7 PXAs were analyzed with a whole
genome microarray of 6.000 large insert clones resulting in a resolution of
at least 1Mb. In each of these cases, the results obtained by chromosomal-
CGH could also be detected by the matrix-CGH experiments. In 3 of the
7 PXAs, additional aberrations were found by matrix-CGH. Imbalances
detected by matrix-CGH were veried by interphase-FISH on tumor tissue
sections. In particular, breakpoints were conrmed in one case with partial
deletions on 9p and 18p. Molecular genetic analysis of selected candidate
genes revealed TP53 mutations in only 3 of 62 (5%) PXAs analyzed. The
CDKN2A, CDKN2B, and p14
ARF
genes on 9p21 did not show homozy-
gous deletion, mutation, promoter hypermethylation, or complete loss of
mRNA expression. None of the tumors showed mutation of the PTCH
or TSC1 genes or amplication of the EGFR, CDK4, or MDM2 genes.
Taken together, our study provides a comprehensive overview of genetic
alterations in PXAs and indicates that chromosome 9 carries one or more
not yet identied tumor suppressor gene(s) with relevance to the molecular
pathogenesis of these tumors.
313. ALTERATIONS OF p53 AND p73 OCCUR
INDEPENDENTLY OF ONE ANOTHER IN ALL GRADES OF
PEDIATRIC DIFFUSE ASTROCYTOMA
S. Ward,
1
B. Suarez-Merino,
1
Y. Ding,
1
R. Hayward,
2
W. Harkness,
2

D. Thompson,
2
K. Phipps,
2
D. Thomas,
3
and T. Warr
1
;
1
Department
of Molecular Neuroscience, Institute of Neurology;
2
Department of
Neurosurgery, Great Ormond Hospital for Sick Children NHS Trust;
3
Division of Neurosurgery, Institute of Neurology, London; UK
The p53 tumor suppressor gene plays an essential role in cell cycle
arrest, response to DNA damage, and apoptosis, and abnormalities of p53
are present in many tumor types. Also, p73 shows structural and func-
tional similarity to p53 and is the potential gene target of 1p36.3 deletion in
human cancer. Inactivation of p53 through mutation, allelic loss, or hyper-
methylation of CpG islands in the promotor region occurs in approximately
30% of all three grades of adult astrocytoma and is strongly associated
with the development of secondary GBM; p73 methylation has also been
reported in malignant astrocytoma in adults. However, the roles of p53
and p73 in pediatric astrocytoma remain equivocal. In this study, we deter-
mined the frequency of p53 mutations by direct sequencing of exons 4 to 7
in 34 pediatric astrocytoma comprising 13 WHO grade II, 3 WHO grade
III, and 18 WHO grade IV tumors. We also investigated the methylation
status of both p53 and p73 using methylation-specic PCR. Mutations of
p53 were present in 2 samples of high-grade astrocytoma only. One grade
III astrocytoma showed a mutation at codon 271 (GTG to GAG, Val to
Glu), while the second mutation was observed at codon 282 (CGG to TGG,
Arg to Trp) in a grade IV tumor. Methylation of p53 was detected in 8
cases (24%) comprising 3 grade II (23%), 1 grade III (33%), and 4 grade
IV (22%) tumors. Eleven (32%) samples showed methylation of p73, com-
prising 6 grade II (46%) and 5 grade IV (27%) astrocytoma. Methylation
of p73 occurred independently of both mutation and methylation of p53.
Overall, alterations of p53 or p73 were present in 58% of tumors. This data
indicates that although mutations of p53 are rare in pediatric astrocytoma,
methylation of the promotor region may offer an alternative mechanism of
p53 inactivation. This contrasts with adult astrocytoma in which mutation
of p53 is much more common than hypermethylation. Similarly, in adult
astrocytoma, methylation of p73 has been observed predominantly in grade
IV tumors and there is no correlation with p53 status. The ndings in the
present study that p73 methylation occurred only in tumors with no altera-
tions of p53 and at similar frequencies in low-grade and malignant tumors
provides further evidence of a different genetic pathogenesis between pedi-
atric and adult astrocytoma.
314. INTERSTITIAL LOSS AND GAIN OF SEQUENCES ON
CHROMOSOME 22 IN MENINGIOMAS WITH NORMAL
KARYOTYPE
A. Prowald,
1
S. Urbschat,
2
S. Wemmert,
1
R. Ketter,
1
E. Meese,
2

K.D. Zang,
3
and W.-I. Steudel
1
;
1
Neurochirurgie, Neuroonkologie,
Universittskliniken des Saarlandes, Homburg/Saar;
2
Institut fur
Humangenetik, Universitt des Saarlandes, Saarbrcken;
3
IGD Saar,
Institut fr genetische Diagnostik, Homburg/Saar; Germany
In nearly half of sporadic low-grade meningiomas, no chromosome
aberration can be detected. In the majority of the other half, chromosome
22 is lost. In higher grade meningiomas, this loss is followed by character-
istic secondary chromosome aberrations. Regarding the molecular ndings
in schwannomas, homozygous loss or mutation of the NF2 gene located on
chromosome 22 was supposed also to be the primary event in meningioma
development. However, in nearly all high-grade but in only a minority of
low-grade meningiomas, the loss of the NF2 protein is observed. Therefore,
the hypothetical combined heterozygous loss of or inactivation of two or
more tumor suppressor genes (at least one of them located on chromosome
22) as well as the homozygous loss of a regulatory gene on chromosome 22
different from NF2 was discussed. In a search for microdeletions or/and
structural recombinations of chromosome 22, we investigated primary cell
cultures of 43 meningiomas by conventional G-banding (26 without, 17
with loss of chromosome 22). Twenty-seven tumors were analyzed with
spectral karyotyping (SKY) and 16 with uorescence in situ hybridiza-
tion (FISH) with DNA probes for the chromosomal regions of 22q11.2,
22q11.23q12.1, 22q12.1, and 22q13.3. SKY analysis conrmed G-banding
data for chromosome 22 and could specify marker chromosomes and
translocations containing material from chromosome(s) 22. Conrming
our assumption, microdeletions on chromosome 22 were detected by FISH
in 6/8 cytogenetically nonaberrant meningiomas. Surprisingly, in 2/8 cases
we observed gains of the 22q13.3 region and in 2/8, gains of the 22q12.1
region. Here we present rst evidence for an uncommon mechanism dur-
ing early meningioma development at least for a meningioma subgroup: (i)
duplication and translocation of sequences from chromosome 22 to differ-
ent chromosomes, (ii) deletion of the original sequences on chromosome 22,
resulting in disomy again (only visible as translocation in metaphase FISH),
and (iii) loss of chromosome 22.
315. CORRELATING GENE EXPRESSION TO DNA COPY
NUMBER IDENTIFIES GENES INVOLVED IN THE
PATHOGENESIS OF HUMAN GLIOBLASTOMA
J. Nigro,
1,2
A. Misra,
1,2
I. Smirnov,
1,2
L. Prestegarden,
6
L. Wanyu,
7

R. Yeh,
3
K. Aldape,
8
S. VandenBerg,
2,4
and B. Feuerstein,
1,2,5
;
1
Cancer
Center and Departments of
2
3
Epidemiology
and Statistics,
4
Pathology, and
5
Laboratory Medicine, University of
California, San Francisco, San Francisco, California, USA;
6
Biomedicine,
University of Bergen, Bergen, Norway;
7
Neurosurgery, University of
Lausanne, Lausanne, Switzerland;
8
M.D. Anderson Cancer Center,
Houston, Texas, USA
Tumor behavior is related to molecular changes that occur during
neoplastic development. In this study, we used expression and genomic
array analysis to reveal molecular proles associated with patient sur-
vival in human glioblastomas. Cases fell into two genetic subgroups with
poorer survivors (2 years) associated with chromosome 7 gain and 10
loss. Expression proles of the same tumors were strongly associated with
patient survival. We correlated these two data sets to explore how gene
expression is inuenced by underlying genomic changes. We found that
gene expression was correlated with DNA copy number, but that loss of
chromosome 10 was a much stronger inuence on global gene expression.
One homeobox gene, Meox2, that is expressed during mesoderm induction
and required for normal muscle development, was strongly associated with
both patient survival and chromosome 10 loss. The expression array data
showed that in cases with chromosome 10 loss, Meox2 was fourfold over-
expressed relative to those where chromosome 10 was intact and greater
than eightfold overexpressed relative to non-neoplastic brain. Expression
array results were conrmed by real-time reverse transcriptase PCR. By
immunohistochemistry, we localized the protein to the nuclei of primary
human glioblastomas, xenografts, and tumor cell lines. We are using bio-
logical and statistical models to identify genes regulated coordinately with
Meox2 expression. The results indicate that molecular approaches can iden-
tify biologically distinct groups of human glioblastomas and genes that may
contribute to the pathogenesis of human gliomas. This work was supported
by the NIH (PO1 NS42927 and CA 85799) and the National Brain Tumor
Foundation.
316. POSSIBLE RELATIONSHIP BETWEEN P27/
KIP.1 AND CYCLIN D1 DEGRADATION AND THE
IMMUNOPROTEASOME IN GLIOBLASTOMA
D. Schiffer,
1,2
M. Piccinini,
3
A. Anselmino,
3
V. Fiano,
2
C. Ghimenti,
2

M. Rinaudo
3
;
1
Neuroscience, University of Turin, Turin;
2
Research
Centre for Neuro-bio-pathology Oncology, Foundation Policlinico
di Monza, Vercelli;
3
Medicine Experimental Oncology, Section
Biochemistry, University of Turin, Turin; Italy
The cell cycle is regulated by the cyclin/kinase complexes with p27/
Kip.1 functioning as CDK inhibitor at the checkpoint G1-S. Cyclin D1 and
p27/Kip.1 are under the control of PKB/Akt pathway, which is a crosspoint
between Ras-MAPK from PTKR and PTEN pathway. Phosphorylated Akt
activates cyclin D1 through AKT/FRAP mTOR and inhibits p27/Kip.1
through AFX/FKHR. In gliomas, cyclin D, E, A, and B1 and CDK4-6-
2 and cdc2 expression increases with malignancy, whereas that of p27/
Kip.1 decreases in parallel with the increase of its F-box targeting protein
Skp2. Among many other proteins, cyclins and p27/Kip.1 are degraded
into the ATP-dependent ubiquitin-proteasome system. Ubiquitin ligation
with proteins targeted for degradation is accomplished by an enzymatic
complex, E1-E2-E3, and the ubiquitinated protein is then substrate for the
26S proteasome, which cleaves the proteins into small peptides by means
of its catalytic core (20S proteasome). The 20S proteasome contains a- and
- constitutive subunits with proteolytic activity. Under cytokine stimula-
tion, constitutive subunits 1, 2, and 5 are replaced by the inducible sub-
units LMP2, MECL-1, and LMP7, transforming the proteasome into the
immunoproteasome. Based on the previous study of cyclins and p27/Kip.1
in a series of gliomas, 18 glioblastomas were studied by immunohistochem-
istry for inducible subunits and evaluated for peptidase activity and subunit
composition by spectrouorimetric assay and Western- immunoblotting.
In malignant gliomas, it has been observed that the inducible subunits are
more expressed, but their proteolytic activity is strongly reduced in com-
parison with controls. Preliminary results indicate that the 26S proteasome
does occur in glioblastomas. Post-translational modications of the 20S
proteasome can be responsible for the discrepancy, and the nding is con-
sistent with the increased expression of cyclin D1 in glioblastomas, but not
with the reduction of p27/Kip, which is a proteasome substrate. It may be
that in glioblastomas the immunoproteasome expression represents a more
adequate way for the degradation of p27/Kip.1.
317. DISSECTING GLIOMA SYSTEMS BY GENOMICS,
PROTEOMICS, AND MODELING
W. Zhang, I. Shmulevich, R. Jiang, C. Mircean, and G. Fuller; Pathology,
M.D. Anderson Cancer Center, Houston, Texas, USA
Cancer systems are overwhelmingly complex. They, like healthy cells,
are regulated by numerous genes and proteins in a highly coordinated fash-
ion. The main difference is that in cancer cells, the rules of regulation have
been altered by events that cause the underlying complex dynamical system
governing cellular activity to behave in an aberrant manner. The complex
nature of a cancer system, with its many unrevealed components and levels
of complexity, calls for a more systematic measurement of gene and pro-
teins and the development of suitable representational models that can ulti-
mately be used to guide cancer researchers in their pursuit of understanding
cancer and nding effective treatment. Using microarray technology, we
have proled gene expression of different grades of gliomas. In addition
to some highly interesting individual markers such as insulin-like growth
factor binding protein 2 (IGFBP2), we have developed a mathematical
model called Probabilistic Boolean Network (PBN) to construct a network
based on expression of 600 genes that are functionally well characterized.
The network has revealed novel relationships among genes, some of which
have subsequently been conrmed by experiments. For example, the PBN
model revealed a relationship between IGFBP2 and NFkappaB, which was
experimentally conrmed. To extend our understanding, we have recently
constructed a reverse-phase protein lysate array with 90 different glioma
tissues and assayed for the expression of 50 signaling proteins. To gain
a quantitative insight, each sample is serially diluted and in triplicate on
the array. Mathematical analyses have revealed key protein expression
and posttranslational modication events during glioma progression. For
example, akt phosphorylation is most dominant in glioblastomas. In sum-
mary, we begin to be able to dissect the glioma systems with genomics,
proteomics, and bioinformatics.
318. TELOMERASE AND ESTROGEN RECEPTOR ROLES IN
GLIOMAS
N. Cruickshanks,
1
A. Shervington,
1
R. Lea,
1
L. Shervington,
1
and
G. Roberts
2
;
1
Biological Sciences, University of Central Lancashire,
Preston;
2
Department of Neurosurgery, Royal Preston Hospital, Preston;
UK
The most common primary brain tumors are glioma. Glioma cells that
respond to chemotherapy have shown a decline in the activity of a specic
enzyme called telomerase. This enzyme is usually only active in undiffer-
entiated cells. However, it has been detected in 94% of neuroblastoma and
100% of oligodendroglioma. Numerous studies have concentrated on the
induction level of hTERT, the major subunit of telomerase in cancer tissue,
but few workers have correlated the level of telomerase with hsp90 and p23
in cancer cells and glioma tissue. Hsp90 and p23 both function as a co-
chaperone for the telomerase. It is proposed here that if there is high consti-
tutive expression level of hTERT, then there may also be a high expression
level of hsp90 and a lower expression level of p23, since this is a cancer
suppressor. Furthermore, a high brain concentration of telomerase protein
may offer an alternative and direct indicator of malignancy. In this study,
the level of telomerase in cell samples has been quantitatively measured
by several methods. Results indicate an elevated constitutive expression
of telomerase in cancerous cell lines with respect to control cells. The cells
lines used in this study namely are the following: U-87-MG (glioblastoma
astrocytoma), 1321N1 (astrocytoma), IPDDC-A2 (astrocytoma; grade
II), GOS-3 (astrocytoma/oligodendroglioma), JCRB0068 (brain, embry-
onic), and NHA (normal astrocytes). Unxed tumor biopsy material will
be obtained from Royal Preston Hospital. Handling of the tissue followed
university code of practice. Total RNA was isolated from biopsy tissues,
primary cells, and cell lines using RNA capture kit (Roche). RNA will be
used to generate cDNA using a Reverse Transcription System (Roche). A
potential application for this work is to use it as a diagnostic tool for glioma
cancer by nding the right marker.
319. HYPERDIPLOIDY DEFINES A DISTINCT CYTOGENETIC
ENTITY OF AGGRESSIVE MENINGIOMAS
R. Ketter,
1
Y.-J. Kim,
2
S. Storck,
3
W.-I. Steudel,
1
K.D. Zang,
4
and
W. Henn
3
;
1
Neurosurgery and Neurooncology and
2
Neuropathology,
Kliniken des Saarlandes, Homburg/Saar;
3
Institute of Human Genetics,
Universitt des Saarlandes, Saarbrcken;
4
Institut fr Genetische
Diagnostik, IGD, Homburg/Saar; Germany
The most common chromosomal aberration found in meningiomas of
all grades is monosomy 22. Progression and recurrence of meningiomas
is usually associated with stronger hypodiploidy, i.e., monosomy of fur-
ther autosomes and, most frequently, heterozygous loss of chromosome 1p.
Rarely, however, hyperdiploid karyotypes occur; the objective of this study
was to explore the cytogenetic and histopathologic patterns as well as the
clinical signicance of hyperdiploidy in meningiomas. A consecutive series
of over 400 meningiomas were cultured in vitro and cytogenetically char-
acterized by using standard banding techniques and, in one structurally
aberrant case, spectral karyotyping (SKY). In patients with hyperdiploid
meningiomas, clinical and histomorphological data as well as results of
long-term postoperative survey were compared with data from patients with
cytogenetically typical meningiomas. We identied a subgroup comprising
about 4% of all meningiomas that do not display the common chromosome
losses but instead a strikingly uniform pattern of hyperdiploidy. Mostly in
the absence of structural chromosome rearrangements, these meningiomas
each have between 49 and 56 chromosomes, with trisomy 12 (14/16 cases),
trisomy 20 (13/16 cases), trisomy 5 (12/16 cases), and trisomy 17 (10/16
cases), along with variable trisomies of all other autosomes except #1, #2,
and #21. Chromosome losses are rare, affecting #22 in 2/16, and #7 and
Y each in only 1/16 cases. Histomorphologically, the hyperdiploid menin-
giomas show intermediate differentiation with patternless growth and/or
microcystic degeneration. However, a loss of chromosome 22 may be asso-
ciated with a persistent brous growth pattern. The proliferative potential
in terms of increased mitotic activity and Ki-67 labeling index is signi-
cantly elevated; all investigated hyperdiploid meningiomas were assigned
to WHO grade II. Fourteen patients in whom tumor resections were deter-
mined to be simpson grade I or II and 2 patients with simpson grade III
could be followed up after tumor extirpation. In 2 patients, recurrences
were documented and 3 patients died during the period of observation.
We conclude that hyperdiploidy constitutes a small but clinically relevant
entity of biologically aggressive, histopathologically atypical meningiomas
(WHO grade II), which are cytogenetically distinguishable from the major-
ity of common-type meningiomas.
320. PHARMACOLOGICAL REVERSAL OF GENE
SILENCING IN MALIGNANT GLIOMA: A WHOLE GENOME
MICROARRAY ANALYSIS
G. Foltz and A. Madan; Neurosurgery, University of Iowa College of
Medicine, Iowa City, Iowa, USA
Promoter methylation and histone modication play a critical role in
transcriptional regulation. We explore the role of histone deacetylation in
aberrant gene silencing in malignant glioma. Histone deacetylase inhibi-
tors induce growth arrest and apoptotic cell death in malignant astrocytes.
We use a whole-genome microarray analysis to identify genes differentially
expressed in malignant astrocytes in response to treatment with Trichosta-
tin A (TSA), a specic inhibitor of histone deacetylase activity. ChIP analy-
sis of histone modications identied specic promoter region alterations in
selected differentially regulated genes. Microarray studies were performed
on 11 primary glioblastoma cell lines (UI series), the T98 and U87 cell lines,
and normal human astrocytes. Three experimental paradigms were used:
(1) a time-course analysis of T98 cells treated with 1 M TSA for 6, 12, 24,
36 and 48 h, (2) a dose-response analysis of T98 cells treated with increas-
ing doses of TSA (300 nM to 5 M), and (3) a threshold analysis requiring
response in 8 of 11 primary lines and U87 cells treated with 1 M TSA for
24 h. Biological replicates were performed independently in triplicate for
each condition. A cohort of TSA regulated genes was selected as an inter-
section of the three experimental paradigms after statistical analysis using
Signicance of Microarrays (SAM) and ANOVA/Q-Value (Storey) with
maximum stringency (q 0.001). GBM specic responses were identied
by comparison to normal human astrocytes treated with 1 M TSA for 24
h. Differentially expressed genes were conrmed with real-time PCR and
evaluated for promoter region histone modication using ChIP analysis
with antibodies to K4-methylation and K9/14 acetylation. Promoter regions
of select genes were also analyzed with bisulte sequencing. Two selected
genes were transfected into T98 and U87 cells resulting in suppression of
cell growth in a colony focus assay. Over 800 genes were upregulated at
least 2-fold across all conditions. Fifty selected genes were conrmed by
real-time PCR. Eight of 10 selected genes had increased acetylation of pro-
moter region K9/14 acetylation, independent of promoter methylation and
K4-methylation status. Two genes demonstrated suppression of cell growth
after plasmid-mediated transfection in U87 and T98 cells. GBM specic
gene expression proles resulting from histone deacetylase inhibition pro-
vides insight into the mechanisms of aberrant transcriptional regulation
during malignant astrocyte transformation. The identication of epigenetic
silenced genes represents a potential entry point for biomarker development
and therapeutic intervention.
321. MOLECULAR CLASSIFICATION OF GLIOMAS BY
COMPARATIVE GENOMIC HYBRIDIZATION ARRAY IS
CORRELATED WITH PROGNOSIS
A. Idbaih,
1
Y. Marie,
2
A. Aurias,
1
P. Pierron,
1
K. Mokhtari,
2
M. Kujas,
2

J. Delattre,
2
M. Sanson,
2
K. Hoang-Xuan,
2
and O. Delattre
1
;
1
Institut
Curie, Unit INSERM U509, Paris;
2
Hpital Piti-Salptrire, Unit
INSERM U495, Paris; France
The purpose of our study was to screen gliomas for genomic imbalances
by the comparative genomic hybridization array technique (CGHa). DNA
from 100 unselected diffuse gliomas were investigated by a genome-wide
array-based CGHa technique (a total of 3342 mapped and amplied BAC
DNA were spotted in an array format on glass, providing an average resolu-
tion of 1 Mb across the human genome). The genomic proles of all tumors
were classied by using clustering software. The results were correlated
with the outcome of the patients. The most frequent alterations were loss
of 1p, 19q, 9p (P16/CDKN2A locus), and 10q; gain of chromosome 7; and
amplication of EGFR. In addition, CGHa detected less well-documented
recurrent abnormalities (loss of 4, gain of 19, amplication of PDGFRa,
MDM4, MDM2 and CDK4 locus). The results were concordant with those
obtained in the same series of tumors with other conventional techniques
(LOH and real-time PCR techniques). Moreover, CGHa allowed us to
detect small interstitial deletions that would be unlikely detected by other
methods, so that we could narrow considerably the candidate region on 1p.
Lastly, gliomas could be classied according to their CGHa genetic prole
into several groups demonstrating distinct clinical outcome. CGHa is a reli-
able and powerful method to investigate genomic imbalances in gliomas,
to narrow candidate regions and identify novel genetic alterations, and to
classify the tumors into distinct prognostic subgroups.
322. ANALYSIS OF MEDIUM THROUGHPUT
QUANTITATIVE LOH IN ANAPLASTIC
OLIGODENDROGLIOMA
R.A. Betensky,
1
E.A. Houseman,
1
B.A. Coull,
1
T. Mikkelson,
2
L.
Tahash,
2
J.G. Cairncross,
3
D.N. Louis,
4
and O. Bgler
2
;
1
Biostatistics,
Harvard School of Public Health, Boston, Massachusetts, USA;
2
Neurosurgery, Henry Ford Hospital, Detroit, Michigan, USA;
3
Foothills
Medical Centre, University of Calgary, Canada
Allelic losses on chromosomes 1p and 19q are known to occur fre-
quently in anaplastic oligodendrogliomas. Current assessment of LOH in
oligodendrogliomas is based on 3-4 distally located CA-repeat polymor-
phism markers. A call of LOH is made if LOH is detected at all infor-
mative markers. We recently used capillary electrophoresis for assessing
LOH at 15 markers on 1p and 4 markers on 19q in 93 tumors. We derived
meaningful thresholds for the quantitative LOH through the use of a latent
mixture model that leveraged the measurements from normal specimens to
gain information about the non-LOH distribution. We applied latent class
analysis to cluster subjects according to patterns of LOH and found three
distinct LOH proles among our samples: a group with LOH at all mark-
ers, a group with moderate LOH, and a group with low LOH. The survival
outcomes of the high-LOH group were signicantly more favorable than
those of the other groups, which were not different from each other. We
used the latent class model to multiply impute values for the noninformative
LOH outcomes and thereby increased the power of the survival analyses.
Last, we developed constrained estimation techniques to enable complete
joint modeling of all 19 markers while avoiding overtting the data. These
techniques can be applied more generally to the analysis of other genomic
assays, including array comparative genomic hybridization (aCGH).
323. IDENTIFICATION OF NOVEL GLIOBLASTOMA TUMOR
SUPPRESSOR GENE CANDIDATES ON CHROMOSOME
10q24-qTER
G. Reifenberger, M. Wolter, B. Khler, and C. Knobbe; Neuropathology,
Heinrich-Heine-University, Duesseldorf, Germany
Allelic losses on chromosome 10 are common genetic alterations in
glioblastomas that are found in approximately 60% to 90% of the cases.
Molecular genetic analyses suggested the presence of up to 3 distinct tumor
suppressor gene loci at 10p, 10q23, and 10q24-qter, respectively. The PTEN
tumor suppressor gene has been identied as the relevant gene on 10q23.
The KLF6 gene at 10p15 had been suggested as another glioblastoma sup-
pressor gene, but does not appear to carry frequent alterations in these
tumors. Thus, the target genes on 10p and 10q24-qter are not known yet.
We performed real time-RT PCR expression analysis of 17 genes located
on 10q24-qter in a series of 34 primary glioblastomas and 12 secondary
glioblastomas to identify novel candidate tumor suppressor genes that are
downregulated in glioblastomas. The genes were either selected from the
NCBI Human Genome Browser (www.ncbi.nlm.nih.gov) or from microar-
ray expression proling data of glioma cell lines treated with the demeth-
ylating agent 5-aza-deoxycytidine and the histone deacetylase inhibitor
trichostatin A. So far, we identied 3 genes (ADD3, EMX2, and OAT) that
show markedly reduced mRNA levels relative to non-neoplastic brain tissue
in substantial fractions of glioblastomas. Furthermore, treatment of glioma
cell lines with 5-aza-deoxycytidine and trichostatin A resulted in increased
expression of these genes, suggesting that their transcriptional downregula-
tion may be due to promoter hypermethylation. These three genes represent
interesting novel tumor suppressor candidates that are presently further
investigated for promoter hypermethylation and coding region mutations.
324. GENES THAT PROMOTE CELL GROWTH ARE
ABERRANTLY EXPRESSED IN PILOCYTIC ASTROCYTOMA
N. Potter,
1
R. Poh,
1
S. Ward,
1
B. Suarez-Merino,
1
W. Harkness,
2
R.
Hayward,
2
D. Thompson,
2
K. Phipps,
2
J. Rees,
1
D. Thomas,
3
and
T. Warr
1
;
1
Institute of Neurology, UCL, London;
2
Department of
Neurosurgery, Great Ormond Street Hospital for Sick Children NHS
Trust, London;
3
Division of Neurosurgery, Institute of Neurology,
London; UK
Astrocytoma are the most common brain tumors occurring in chil-
dren. The majority of these are low-grade pilocytic (grade I) and diffuse
(grade II) astrocytoma comprising 80% of cases. Little is known about the
genetic basis underlying the development of pediatric astrocytoma. Previous
cytogenetic studies have revealed that the majority of low-grade pediatric
astrocytoma appear to be karyotypically normal. It is therefore possible
that abnormal gene expression rather than chromosomal abnormalities are
involved in the development of these tumors. The oligonucleotide Affyme-
trix Human Genome U133 Array representing 33,000 genes was used to
generate expression proles of 14 grade I pediatric astrocytoma biopsy
samples and three normal brain controls. Genespring version 6.1 was used
for array data analysis including the completion of 1-way ANOVA statisti-
cal tests. In total 1340 genes were differentially expressed in tumor samples
compared to normal brain controls; 1063 were upregulated and 277 were
downregulated. Those genes overexpressed included components of the Wnt
signaling pathway, Wnt5A, FDZ6, PKC, CKIa, LRP6, cyclin D1, v-jun,
and TCF, as well as genes associated with the Notch and TGF-b signaling
pathways, Bmi-1, JAG1, BMP2, Hey-1, Id4, and Bcl-2. Growth promoters
PDGFRa, PDGFC, and FGF2 were also upregulated. The downregulation
of SMAD7 and SMURF1 may increase TGF-b pathway signalling, and the
downregulation of tumor suppressor genes p57, p19, and MTUS1 may also
contribute to tumor growth. In contrast, the tumor suppressor gene p53 was
signicantly upregulated. Differential gene expression in pilocytic astrocy-
toma indicates increased activity of pathways involved in cell growth.
325. GENE EXPRESSION PROFILING OF GLIOMAS
J. Takahashi,
1
K. Koizumi,
2
S. Saito,
2
S. Oba,
3
S. Ishii,
3
K. Kato,
2
N.
Hashimoto,
1
and M. Shirahata
1
;
1
Neurosurgery, Kyoto University,
Kyoto;
2
Functional Genomics, Research Institute, Osaka Medical
Center, Osaka; Theoretical Life Science,
3
Nara Institute of Science and
Technology, Nara; Japan
We analyzed the expression level of 3456 genes in 109 glioma using
adaptor-tagged competitive PCR(ATAC-PCR), a high-throughput reverse
transcription-PCR technique. The purpose of this study is to investigate
the molecular features of glioma through a large-scale gene expression
proling. A total of 109 gliomas specimens including 80 glioblastomas,
10 anaplastic astrocytomas, 12 diffuse astrocytomas, and 7 anaplastic oli-
godendrogliomas were obtained from surgical resection. We rst surveyed
the genes actually expressed in glioma using expressed sequence tag (EST)
sequencing. A 3-end cDNA library was constructed by using a mixture
of RNA from 12 gliomas, and we randomly selected 3036 genes from this
EST collection. We then designed PCR primers for 3456 genes including
420 genes known to be expressed in glioma from previous literature. The
expression level of these genes in sample RNAs derived from 109 gliomas
was assayed by the ATAC-PCR technique. ATAC-PCR is an advanced
version of quantitative competitive PCR, characterized by the addition of
unique adaptors for different cDNAs, measuring the relative expression of
samples against the control. In this assay, using seven adaptors, 4 samples
and 3 known amounts of controls were processed in a single reaction. The
PCR amplication was performed using an adaptor-primer, the sequence
of which was from the common part of adaptors, and a primer specic
to the gene of interest, the sequence of which was included in the 3 end
fragment. Amplied fragments were separated by denaturing polyacryl-
amide gel electrophoresis, and the amount of fragments was measured by
an automated sequencer. We analyzed the expression levels of 3456 genes
in 109 gliomas. The unsupervised hierarchical cluster analysis revealed that
different types and grades of glioma had a unique gene expression pattern
of a specic group of genes. In addition, glioblastomas were divided into
molecular groups, each of which possessed a distinct gene expression sig-
nature. Our gene expression study showed that each histological type and
grade of glioma has a distinct gene expression signature. Our results may
lead to molecular classication of gliomas.
326. NAVIGATING GLOBAL TRANSCRIPTOMICS TO
THERAPEUTIC TARGETS ON INVASIVE GLIOMA CELLS
J. Rennert,
1
T. Demuth,
1
D. Hoelzinger,
1
L. Reavie,
1
L. Mariani,
2
K.
Pan,
3
C. Lih,
3
R. Lin,
3
S. Cohen,
3
and M. Berens
3
;
1
Department of
Neurogenomics, Translational Genomics Research Institute, Phoenix,
Arizona, USA;
2
Switzerland;
3
Department of Genetics, Stanford University School of
Medicine, Stanford, USA
Gliomas are the most common primary brain tumors, they exhibit
highly invasive behavior, which is a major cause of the morbidity and
prompt lethality of the disease. A better understanding of the determinants
of glioma migration and invasion may explain the ubiquitous recurrence
of glioblastoma and may reveal novel therapeutic targets aimed at inva-
sive cells. Motile and stationary cell populations from seven human glioma
cell lines and three primary glioblastoma cultures were isolated from a
monolayer radial migration assay for gene expression analysis using oligo-
nucleotide microarrays. Differentially expressed genes between these two
populations across all cell lines were identied using a pattern recognition
approach that integrates a priori knowledge in conjunction with expres-
sion data as implemented in GABRIEL (Genetic Analysis by Rules Incor-
porating Expert Logic), a Web-based application designed for rule-based
analysis) (Proc. Natl. Acad. Sci. USA 99, 2118, 2002). Principal component
analysis (PCA) is a method for reducing high dimensional data into a few
components that represent the majority of variation within the data. PCA
of the differential expression data revealed two discriminating patterns, a
global downregulation prole and a global upregulation prole. Two genes
(AK098354 and Cyr61 [cystein rich 61]) following these proles were used
in GABRIELs proband rule-based function to nd subsets of genes with
similar expression patterns. Differential expression of eight candidates
(four from each subset) was validated by QRT-PCR. Of these eight, Cyr61
and CTGF (connective tissue growth factor) are secreted extracellular pro-
teins in the CCN family of growth factors, (Cyr61, CTGF, and Nov) whose
properties include tumorigenesis and cell migration. Immunouorescence
conrmed increased protein expression between motile and stationary cells
in a migration assay. Immunochemistry on glioma invasion tissue microar-
rays revealed expression of Cyr61 and CTGF in association with invasion.
siRNA knockdown of Cyr61 showed a reduction in mRNA expression. Its
effect on motility will be examined in a migration assay. These ndings pro-
vide strong evidence that Cyr61 and CTGF play important roles in glioma
invasion potentially leading to new anti-invasion therapies.
327. THE TRANSCRIPTION FACTOR FOXG1 IN GLIOMAS
A.B. Lassman,
1
Q. Wang,
2
J. Massague,
2
and E.C. Holland
1,2,3
;
1
Neurology,
2
Cancer Biology & Genetics, and
3
Surgery (Neurosurgery),
Memorial Sloan-Kettering Cancer Center, New York, New York, USA
Foxg1 is a winged helix/forkhead transcription factor previously called
brain factor 1 (BF-1). It is critical for the normal development of the brain
as demonstrated by the lack of cerebral hemispheres in knockout mice. In
addition, the chicken ortholog (Qin) is an oncogene that was originally
isolated from avian sarcoma virus 31; it both transforms cultured chicken
embryonic broblasts and induces brosarcomas in birds. Evidence also
suggests that Foxg1 contributes to deregulated proliferation by repressing
transcription of the tumor suppressor p21CIP. Finally, Foxg1 is overex-
pressed in low- and high-grade human gliomas as well as the U87MG glio-
blastoma cell line relative to normal human brain. As many parallels exist
between normal brain development and gliomagenesis, these features of
Foxg1 function imply a role in glioma biology not described previously.
We modeled the pathways driving human glioma growth both in vitro and
in vivo to explore the role of Foxg1 in glioma formation. We rst trans-
formed primary mouse glial progenitors in culture with activated forms of
Ras, Akt, Ras Akt, or PDGF by somatic cell gene transfer of the various
oncogenes. We then performed anti-Foxg1 Western blot analysis on protein
lysates from the transformed glia. We also pharmacologically silenced Ras
signaling in the Ras transformed glia with a MEK inhibitor to analyze the
impact on Foxg1 expression. We then used our established mouse glioma
models, including astrocytomas by Ras or RasAkt and oligodendroglio-
mas induced by PDGF, to evaluate the impact on Foxg1 expression in vivo.
Finally, we tested the ability of Foxg1 to induce gliomas both alone and
combined with other oncogenic abnormalities. Glia transformed by Ras
and RasAkt exhibited elevated Foxg1 expression. Pharmacologic inhibi-
tion of Ras signaling reduced Foxg1 expression. These results suggest that
Ras activity induces Foxg1 expression or selects cells expressing Foxg1.
Experiments to evaluate the impact of Ras, Akt, and PDGF on Foxg1 in
modeled gliomas in vivo are ongoing as are experiments to determine the
ability of Foxg1 to cause gliomas. The forkhead transcription factor Foxg1
is critical for normal brain development and is overexpressed in both low-
and high-grade human gliomas. By modeling the pathways driving human
glioma growth, we demonstrated that Ras signaling may be particularly
important for the expression of Foxg1 in glia. We are validating these
results in vivo with mouse glioma models and are also testing the glioma-
genic capacity of Foxg1. The project described was made possible in part
by grant number CA009512 from the National Cancer Institute and by the
America Brain Tumor Association.
328. IDENTIFICATION OF PROTEIN MARKERS FOR BRAIN
CANCER USING EXPRESSION PROTEOMICS
A. Khalil,
1,4
O. Persson,
2
L. Salford,
2
B. Widegren,
3
and P. James
1
;
1
Department of Protein Technology;
2
The Rausing Laboratory, and
3
Division of Tumor Immunology, Lund
University, Sweden;
4
Department of Protein Research, Mubarak City for
Science, Alexandria, Egypt
Gliomas comprise nearly one-half of primary brain tumors and one-
fth of all primary spinal cord tumors. Combined, grade III and IV gliomas
represent about 40% of all primary brain tumors in patients aged 40 to 49
years, and 60% in patients older than 60 years. In most clinical series, grade
III tumors comprise approximately 10% and grade IV 90% of the total
number of high-grade, malignant primary brain tumors. We have carried
out DIGE (Difference gel electrophoresis) analysis on glioma biopsies. The
DIGE technology enables different samples to be run on the same gel by
pre-labeling the samples with three different cyanine dyes. Each sample
is covalently labeled with a different dye from mass and charge-matched
set of uorescent CyDyes, cyanine 2 (Cy2), cyanine 3 (Cy3), and cyanine
5 (Cy5). Effectively, gels can be standardized by using one sample as an
internal standard (pool sample). The pool sample is prepared by mixing
equal amounts of protein from each individual homogenate. Proteomics
promises the discovery of biomarkers and tumor markers for early detec-
tion and diagnosis, novel protein-based drug targets for anticancer therapy,
and new end points for the assessment of therapeutic efcacy and toxicity.
The focus is using the DIGE 2D system to enable large-scale analyses of
samples to generate data sets that can be used to differentiate between the
various brain cancer stages and types. Fifty human brain tissues were run
in duplicate, together with an internal pool sample on each gel. Protein was
extracted with buffer I (2% ASB-16, 8 M urea, 5 mM magnesium acetate,
20 mM Tris-base) and rehydrated in buffer II (4% CHAPS, 7 M urea, 2 M
thiourea, 5 mM magnesium acetate, 1.2 mM Destreak, 1% IPG buffer, 20
mM Tris-base pH 8.5). The standard sample is ideally a pool comprising
equal amounts of each of the 50 brain tissues being compared. The 150
images were acquired, and then spot detection and alignment was carried
out using the Decyder software. In addition, the gels for picking protein
spots were run. The spot cut list was generated covering proteins whose
expression levels were changing, as well as set of marker proteins that were
constant, throughout the DIGE experiment. The proteins of interest were
automatically picked, in-gel digested, and then nally ngerprinted by
MALDI-TOF. Reproducible DIGE spot maps of brain tumor proteins were
obtained. The protein spots were occasionally distributed over the whole
gel but more concentrated in the pH range 47. The protein analyses gave
high condence identications for 118 spots cut from the preparative gel.
One main protein that was clearly dramatically upregulated was astrocyte
glial brillary acidic protein. This marker used by histologists to deter-
mine whether a tumor is a glioma or not. A low-sulfate fragment of the
chondroitin sulfate proteoglycan called brevican was also identied which
known to be excreted by tumor cells of patients with brain tumors. A series
of proteins were identied in this preliminary analysis that include known
generic tumor markers such as 78 kDa glucose-regulated protein, annexin I,
and several carbonic anhydrase isoforms. Other proteins that were thought
to be tissue-specic markers were found, and a number of interesting nds
will be presented and further discussed. The day will come when people
will be screened for hundreds of diseases through a simple blood test if our
vision is fullled. Through proteomics, conditions like human cancer will
be diagnosed at a stage early enough so that there will be a good chance they
can be treated and cured.
329. IDENTIFICATION OF A NOVEL HOMOZYGOUS
DELETION REGION AT 6Q23.1 IN MEDULLOBLASTOMAS
USING HIGH-RESOLUTION ARRAY CGH ANALYSIS
H. Ng,
1
A. Hui,
1
H. Takano,
2
K. Lo,
1
W. Kuo,
2
C. Lam,
1
C. Tong,
1

C. Chang,
1
and J. Gray
2
;
1
Department of Anatomical and Cellular
Pathology, The Chinese University of Hong Kong, Hong Kong, China;
2
Laboratory Medicine, University of California, San Francisco,
California, USA
We used a high-resolution array comparative genomic hybridization
analysis for whole-genome analysis of medulloblastomas. Although there
have been many genomic surveys of medulloblastomas in the literature, such
an approach has not been performed before. We identied several consistent
chromosomal aberrations. These included consistent chromosomal gains of
2p23-p25 (52.63%), 7 (57.89%), 9q34 (47.37%), and 17q11-q25 (89.47%),
as well as losses of 3q25.33-q26.32 (57.9%), 4q31-33 (42.1%), 6q22-27
(57.9%), 8p21.3-23 (78.95%), 10q22-26 (57.9%), 16q22-q24 (63.16%),
and 17p13 (31.6%). This information will lead to a better understanding
of medulloblastoma tumorigenesis and identify new molecular markers for
therapeutic intervention and drug discovery. One of the most notable nd-
ings is homozygous deletion on chromosome 6q23. Homozygous deletion
of this region was found on cell line DAOY, whereas copy loss was detected
on 30% primary medulloblastomas. Further study using 30 pairs of STS
markers conned a 0.887 Mb minimal region of homozygous deletion at
6q23.1 which was anked by markers SHGC-14149 (6q22.33) and SHGC-
110551 (6q23.1). Quantitative RT-PCR analysis showed complete loss of
expression of 2 genes located at 6q23.1, AK091351 (hypothetical protein
FLJ34032) and KIAA1913, on cell line DAOY. mRNA level of these genes
was reduced in cell lines D283 and D384, as well as 50% (AK091351) and
70% (KIAA1913) of 10 primary tumors. Frequent detection of reduced
expression of AK091351 and KIAA1913 implicated that these 2 genes may
play a critical role in medulloblastoma tumorigenesis.
330. MOLECULAR CLASSIFICATION OF HUMAN GLIOMAS
USING MATRIX-BASED COMPARATIVE GENOMIC
HYBRIDIZATION
P. Roerig,
1
M. Nessling,
2
B. Radlwimmer,
2
S. Joos,
2
G. Wrobel,
2
C.
Schwaenen,
3
G. Reifenberger,
1
and P. Lichter
2
;
1
Neuropathology,
Heinrich-Heine-University, Duesseldorf;
2
Division of Molecular
Genetics, Deutsches Krebsforschungszentrum, Heidelberg;
3
Department
of Internal Medicine, University of Ulm, Ulm; Germany
Gliomas are morphologically, biologically, and clinically heterogeneous
brain tumors whose classication is based on histological features. How-
ever, evidence is increasing that the different glioma types are associated
with distinct genetic aberrations, which may provide useful information
for tumor classication as well as prediction of prognosis. To facilitate
the molecular classication of gliomas, we established a microarray that
consists of genomic clones representing tumor suppressor genes, proto-
oncogenes and chromosomal regions frequently gained or lost in gliomas,
as well as reference clones distributed evenly throughout the genome in
approximately 15 Mbp intervals. These microarrays were used for matrix-
based comparative genomic hybridization (matrix CGH) analysis of 70
gliomas. Matrix CGH results were validated by molecular genetic analyses
of candidate genes, loss of heterozygosity studies, and chromosomal CGH.
Our results indicate that matrix CGH allows for the sensitive and specic
detection of gene amplications, as well as low-level copy number gains and
losses in gliomas. Furthermore, molecular classication based on matrix
CGH data closely paralleled histological classication, e.g., enabled clini-
cally important differential diagnoses, such as diffuse astrocytoma versus
oligodendroglioma, anaplastic astrocytoma versus anaplastic oligoden-
droglioma, anaplastic oligodendroglioma versus glioblastoma, as well as
primary versus secondary glioblastoma. Thus, matrix CGH is a powerful
technique that allows for an automated genomic proling of gliomas and
represents a promising tool for their molecular classication.
331. DIFFERENTIAL PROTEIN EXPRESSION ANALYSIS OF
TRANSFORMING GROWTH FACTOR-BETA (TGF-BETA)
SIGNALING AND THE IMPACT OF A SPECIFIC LOW-
MOLECULAR-WEIGHT TGF-BETA INHIBITOR IN HUMAN
MALIGNANT GLIOMA CELL LINES
O. Alzate,
1
C. Osorio,
1
M. Herbstreith,
1
M. Hjelmeland,
2
R. Buechler,
1

M. Ehlers,
1
and J. Rich
3
; Departments of
1
Neurobiology,
2
Surgery, and
3
Medicine, Duke University Medical Center, Durham, North Carolina,
USA
Malignant gliomas remain among the most lethal forms of cancer, with
resistance to radiation and chemotherapy. The identication and targeting
of pathways critical to the phenotype of cancers offer new hopes in the treat-
ment of many patients. The multifunctional cytokine transforming growth
factor- (TGF) is expressed by grade III/IV gliomas and promotes tumor
angiogenesis, invasion, and immune escape. We have recently demonstrated
that a novel low-molecular-weight inhibitor of type I TGF receptor kinase
activity, SB-431542, blocks TGF signal transduction and TGF -mediated
expression of VEGF and invasion. We have noted that human glioma cell
lines respond differentially to both TGF and SB-431542. We now seek
to determine markers of cellular response to SB-431542 through protein
expression differences using two-dimensional differential in gel electro-
phoresis (2D-DIGE) proteomics. This is a powerful technique that allows
for the identication of protein expressional changes between control and
target samples. We calculated the relative expression of proteins undergo-
ing different treatments with DeCyder software (Amersham), designed for
analysis in DIGE proteomics, assuming a threshold of 1.67-fold (2 stan-
dard deviations). We treated a human glioma cell line that is responsive to
SB-431542 with vehicle control, SB-431542 (1 M), TGF (100 pM), or
the combination of these two agents. We found that SB-431542 modestly
impacted protein expression when administered alone, whereas TGF sig-
nicantly induced the expression of 11 proteins and repressed the levels
of 5 proteins. Of these proteins, SB-431542 treatment blocked the TGF-
induced expression of 5 proteins. The identities of these proteins are cur-
rently under conrmation and validation. In addition, these studies will be
extended to other glioma cell lines with variable responses to both TGF
and SB-431542. These results suggest that we can identify potential mark-
ers of response to low-molecular-weight signal transduction inhibitors as
well as discover proteins downstream of specic signal transduction path-
ways both dependent and independent of specic kinases. This work was
supported in part by funds from the Pediatric Brain Tumor Foundation of
the United States, Accelerate Brain Cancer Cure, and NIH grant NS047409
(J.N.R.). J.N.R. is a Damon Runyon-Lilly Clinical Investigator and a Sid-
ney Kimmel Cancer Foundation Scholar.
332. PSEUDOPODIAL PROTEOMICS OF GLIOMA CELLS
REVEAL THE SIGNALING AND ENERGY PATHWAYS
ACTIVATED IN MIGRATION
M.E. Beckner,
1
X. Chen,
3
J. An,
3
B.W. Day,
3
and I.F. Pollack
2
;
1
Pathology,
2
Neurological Surgery, and
3
The Genomics and Proteomics
Core Laboratories, University of Pittsburgh, Pittsburgh, Pennsylvania,
USA
The invasive migration of malignant gliomas (astrocytomas) leads to
treatment failure. In vitro, U87 glioma cells exhibited impressive migration.
Although LN229, LN18, U373, C6, and T98G glioma cells migrated to
hepatocyte growth factor (HGF) with fetal bovine serum (FBS) present, U87
cells migrated at comparable levels with only FBS present under normoxia
and hypoxia. All of the migratory glioma cell types expressed Met. Inva-
sive cell migration is initiated by extension of pseudopodia into interstitial
spaces. In this study, U87 cells formed pseudopodia in vitro as cells pushed
through 3-m pores of polycarbonate membranes. Harvesting pseudopodia
in a novel 2-step method provided material for proteomic analysis. Differ-
ences in the protein proles of pseudopodia and whole cells were found by
using differential gel electrophoresis (DIGE) and immunoblotting. Proteins
from 2-dimensional (2D) gels were identied by peptide mass ngerprinting
analysis (PMF) using mass spectrometry. For DIGE, lysates of pseudopodia
and whole cells were each labeled with electrophilic forms of uorescent
dyes, Cy3 or Cy5, and analyzed as mixtures. Analysis was repeated with
reciprocal labeling. Differences in protein distributions were detected by
manual inspection and computer analysis. Pseudopodial proteins in Coo-
massie-stained 2D gels included isoforms of glycolytic enzymes as the
largest group, 7 of 24 proteins. PMF analysis of DIGE gels demonstrated
increased isoforms of annexin (Anx) I, AnxII, enolase, pyruvate kinase, and
aldolase, and decreased mitochondrial manganese superoxide dismutase
and transketolase in pseudopodia. Specic antibodies showed localization
of the HGF a chain (activated form) to pseudopodia and increased pseu-
dopodial Met, actin, and total AnxI (Beckner et al., Lab Invest, in press).
Phosphorylated epidermal growth factor receptor was also increased in
pseudopodia. The proteome of U87 glioma cell pseudopodia includes acti-
vated signaling pathways for migration growth factors/cytokines, potential
downstream targets, and components of the glycolytic pathway to energize
the cytoskeleton. Pseudopodia of other glioma cell lines are being analyzed
for comparison. Identication of pseudopodial proteins offers targets for
suppression of tumor invasion. This work was supported by The Nick Eric
Wichman Foundation, Ellicott City, Maryland, USA, and The Pittsburgh
Foundations Walter L. Copeland Fund for Cranial Research, Pittsburgh,
Pennsylvania, USA.
333. MICRO- GENOMICS OF SCHWANN CELLS IN
PERIPHERAL NERVE SHEATH TUMORS (PNST)
A. Pandita,
1
L. Bereskin,
1
J. Wiley,
1
Z. Karim,
1
A. Okhawat,
1
P.
Shannon,
1
and A. Guha
1,2
;
1
Neurosurgery Division, Toronto Western
Hospital;
2
Neurosurgery Division, Labatts Brain Tumor Centre, HSC,
Toronto, Canada
Neurobroma (nb) subtypes have varying biology with dermal nbs
remaining benign, plexiform nbs having an ~0% risk of becoming malig-
nant termed MPNST. Although consisting of many cell types, Schwann
cells are the primary transformed cells in all subtypes of nbs. Schwann cell
transformation is a result of common bi-allelic inactivation of Nf1 and neu-
robromin expression, in addition to alterations at the genomic level which
likely confer their varying biological properties. Limited conventional CGH
studies on nbs have not yet led to an understanding of these additional
genetic alterations in the Schwann cells of these nb subtypes. Schwann and
endothelial (control) cells were isolated using laser capture microdissection
to create a high-resolution genetic alteration map using array-CGH analysis
on BAC and cDNA arrays. In total to date, 8 plexiform nbs and 8 MPNSTs
have been analyzed. Genetic losses were more prevalent in the plexiform
nbs, compared to MPNSTs, where gains were more common. Loss of
regions on chromosomes 1, 2, 10, 13, 17, and 18 were common in both
benign and malignant tumors. Gains on chromosomal arms 4q, 5p, 6q, 8q,
10q, 11q, 13q, and 17q were commonly found in MPNSTs. Genes located
in regions lost in both plexiform nbs and MPNSTs are those potentially
involved in initiating nbs. Similarly, regions of gain found in MPNSTs
are likely involved in progression from plexiform nbs to MPNSTs. These
array-CGH results will be conrmed using FISH and/or real-time PCR.
FISH analysis using BAC probes for the Nf1 gene corroborated the nd-
ings of the array analysis. Currently, we are utilizing the available NCBI
databases to screen for potential novel oncogenes and tumor suppressor
genes that would be present in these chromosomal regions. These candidate
genes will be further utilized to screen for the frequency of the alteration in
a larger cohort of tumors. Analysis of dermal nbs is ongoing.
334. REAL-TIME QUANTITATIVE PCR REVEALS LOSS
AT 22q12. 3-13. 3 IN TWO THIRDS OF PEDIATRIC
EPENDYMOMA AND DEFINES A MINIMUM DELETION OF
3.4MB
B. Suarez-Merino,
1
R. Hayward,
2
W. Harkness,
2
D. Thompson,
2

K. Phipps,
2
D. Thomas,
3
and T. Warr
1
;
1
Department of Molecular
Neuroscience, Institute of Neurology, London;
2
Department of
Neurosurgery, Great Ormond Street Hospital for Sick Children NHS
Trust, London;
3
Institute of Neurology, Division of Neurosurgery,
London; UK
Ependymomas are glial cellderived tumors which arise from the epen-
dymal lining of the ventricular system of the central nervous system. They
manifest preferentially in childhood, where they account for up to 10%
of intracranial tumors. At present, the genetic events that contribute to
the pathogenesis of this pediatric malignancy remain essentially unknown,
with up to 50% of tumors appearing karyotypically normal. One of the
most common chromosomal aberrations reported in intracranial ependy-
moma is monosomy of 22 or structural abnormalities of 22q, suggesting
the presence of a tumor suppressor gene on that chromosome. Previously,
by expression microarray analysis and real-time quantitative PCR (Q-PCR)
we identied an interstitial deletion at 22q12.3-13.3 in 6 pediatric samples.
In this study we used Q-PCR based deletion analysis to investigate seven
genes mapping to 22q11.3-22q13.3 in a series of 34 pediatric ependymoma
and 32 normal blood controls. The seven genes analyzed were phospha-
tidylinositol 4-kinase (PIK4CA) mapping to 22q11.21, FBX7 (22q12.3),
which may be implicated in phosphorylation-dependent ubiquitination, the
RNA binding protein RBM9 (22q12.3), MFNG (22q13.1), which may be
involved in the Notch pathway, the -catenin interacting protein coding gene
C22orf2 (22q13.1), the chromobox homolog CBX7 (22q13.1), and the SET
binding factor SBF1 (22q13.3). Overall, we observed loss of 22q in 65% of
cases, higher than previously reported in pediatric ependymoma using other
methodologies. The most common region of loss maps between loci RBM9
and CBX7, spanning 3.4 Mb of chromosome 22. Mutation analysis of two
candidate genes mapping to this region (C22orf2 and CBX7) did not reveal
any mutations. Further analysis of the promoter region of CBX7 in tumors
showing underexpression of the CBX7 transcript but no allele loss revealed
that this promoter is not methylated and therefore other mechanisms, such
as histone deacetylation, may be responsible for the silencing of the CBX7
gene in our cohort. Our results provide further evidence that one or more
tumor suppressor genes critical for ependymoma development are located
at 22q12.3-13.1. Further analysis of this region is necessary to identify
candidate transcripts.
335. WHOLE GENOME CDNA MICROARRAY ANALYSIS
OF GLIOMAS: A SUBSET OF GENES DIFFERENTIATES
INVASIVE FROM LESS OR NON-INVASIVE LOW- GRADE
LESIONS
L. Mariani,
1
M. Primig,
2
C. Wiederkehr,
2
and M. Schobesberger
1
;
1
Universittsklinik fr Neurochirurgie, Inselspital, Bern;
2
Swiss Institute
of Bioinformatics, Biozentrum, Basel; Switzerland
Diffuse gliomas WHO grade II-IV are generally not amenable to cure.
Their ability to invade the surrounding normal parenchyma diffusely and
eventually regrow the tumor bulk is unique among cancer types and is prob-
ably the main obstacle for therapeutic intervention. The invasive potential
of these tumors is proportional to the tumor grade. However, magnetic
resonance imaging (MRI) and surgery can reveal striking differences in the
invasion of the brain in tumors sharing the same histopathological charac-
teristics and grade, e.g., in WHO grade II gliomas. The aim of this large-
scale microarray study was to identify genes differentiating invasive from
less- or non-invasive tumors. Tissue samples from 18 gliomas (WHO grades
I to IV), 2 brain metastases, and 4 normal brains were collected during
surgery and immediately transferred into RNA later for storage. Tumors
were graded for their invasive behavior according to MRI (T2-weighted and
FLAIR-sequence). Total RNA was isolated from the samples and processed
according to standard protocols for use in the GeneChip Human Genome
U133 Plus 2.0 Array from Affymetrix which allows analysis of the expres-
sion level of over 47,000 transcripts and variants. The data were analyzed
by hierarchical and two-way clustering algorithms. Five-hundred eight
genes with a maximum intensity 5 across all the samples (19,572 genes)
and a P value 0.01 after a permutation test were identied and selected.
These probesets allowed clustering of the samples according to histological
diagnosis and WHO grade of malignancy. Invasive and non- or less-invasive
low-grade gliomas could be differentiated by hierarchical and coupled two-
way clustering. Whole-genome expression analysis of low-grade gliomas
identies subsets of genes differentially expressed in highly and non- or
less invasive tumors.
336. FUNCTIONAL ASSESSMENT OF GBM-DERIVED
COMPOSITE BAC CLONES IDENTIFIED USING END
SEQUENCE PROFILING
G. Hodgson,
1
S. Volik,
2
and C. Collins
2
;
1
Neurosurgery;
2
University
of California San Francisco, Cancer Research Institute, San Francisco,
California, USA
End sequence proling (ESP) is a novel technique that reveals struc-
tural alterations in tumor genomes such as translocations, inversions, dele-
tions, and amplications. To accomplish this, both ends of a number of
BAC clones from a tumor genomederived BAC library are sequenced and
mapped onto the assembled normal human genome sequence. Recent ESP
studies on breast, brain, and prostate tumors and cell lines have identied a
number of structurally aberrant genomic clones. While the functional sig-
nicance of these clones remains to be determined, one provocative hypoth-
esis is that they encode novel composite transcripts important for tumor
development, akin to the BCR-ABL fusion gene. Indeed, fusion transcripts
have been identied by transcript ESP in breast cancer cell line MCF7. To
test whether glioblastoma (GBM)-derived composite BACs are functionally
signicant for GBM formation, we are testing their effects on prolifera-
tion, apoptosis, and motility when transfected into immortalized human
astrocytes and brain tumor cell lines. Phenotypic alterations in response to
transfection are being measured by using a combination of ow cytometry
and high-content automated imaging microscopy. A total of 5 BACs are
currently under investigation, each of which has been draft sequenced and
ngerprinted in respective collaborations with the Joint Genome Institute
and the Genome Sciences Centre at UBC to conrm their composite nature.
Results from these studies will be presented at the conference. These studies
take an important rst step toward discovery of novel tumorigenic mecha-
nisms in brain cancer and potential development of tumor specic markers
and therapeutics.
337. DNA HYPERMETHYLATION OF MULTIPLE
TUMOR SUPPRESSOR GENES IN DIFFERENT TYPES OF
MENINGIOMAS
J. Kim, H. Kim, and Y. Shim; Department of Neurological Surgery,
Asian Medical Center, Seoul, Korea
Aberrant methylation of CpG islands in promoter of human genes is
known as an alternative mechanism of gene silencing that contributes to
tumorigenesis in various human tumors. We have examined methylation
status of 5 tumor suppressor genes in 81 human meningiomas (61 benign,
12 atypical, and 8 malignant meningiomas) by methylation-specic poly-
merase chain reaction to determine roles for DNA methylation during
transformation to meningioma. Five tumor suppressor genes including p16,
p14, RB, RASSF1A, and E-cadherin showed different prole of methyla-
tion status on CpG islands in their promoter regions depending on stages of
meningioma. Methylation frequencies of tumor suppressor genes in benign,
atypical and malignant meningiomas were 42.6%, 66.7%, and 50.0% for
p16; 16.4%, 50.0%, and 25.0% for p14; 42.6%, 41.7%, and 37.5% for
RB; 1.0%, 8.0%, and 12.5% for RASSF1A; 41.0%, 66.7%, and 12.5%
for E-cadherin, respectively. Except RASSF1A, other tumor suppressor
genes showed relatively high methylation status in meningioma. Forty-six of
61 (75.4%) benign meningiomas, 7 out of 8 (87.5%) malignant meningio-
mas, and all 12 cases (100%) of atypical meningiomas showed hypermeth-
ylation in at least one of the test genes. Surprisingly, DNA was methylated
more frequently in atypical meningiomas than in any other tumor stages:
66.7% for p16, 50% for p14, 41.7% for RB, and 66.7% for E-cadherin.
Our results suggest that DNA methylation is a frequent and an early event
in tumorigenesis of meningiomas, and it appears to be a major molecular
mechanism for meningiomas, especially for atypical type.
338. THE P15INK4B/P16INK4A/RB1 PATHWAY IS
FREQUENTLY DEREGULATED IN HUMAN PITUITARY
ADENOMAS
A. Ogino, Y. Katayama, A. Yoshino, T. Watanabe, T. Fukushima, T.
Yokoyama, C. Komine, and T. Ota; Neurosurgery, Nihon University
School of Medicine, Tokyo, Japan
Pituitary adenomas are common benign intracranial neoplasms. How-
ever, their tumorigenesis is not yet clearly dened. Inactivation of genes
involved in the negative cell cycle regulatory p15
INK4b
-p16
INK4a
-cyclin D/
CDK4-RB1mediated pathway (RB1 pathway) is one of the most com-
mon and important mechanisms in the growth advantage of tumor cells.
Recently, much attention has been focused on the importance of alternative
mechanisms of gene inactivation, particularly promoter hypermethylation
in the transcriptional silencing of such tumor suppressor genes. Based on
the rare occurrence of inactivation by gene mutations and deletions of the
RB1 pathway in pituitary adenomas, we investigated the deregulation of the
RB1 pathway in 42 sporadic human pituitary adenomas, especially focusing
on the methylation status of this pathway as determined by a methylation-
specic PCR assay. Homozygous deletion of the p15
INK4b
or p16
INK4a
gene
was detected in one adenoma each. Amplication of the CDK4 gene was not
apparent in any of the pituitary adenomas presently examined. Promoter
hypermethylation of the p15
INK4b
, p16
INK4a
, and RB1 genes was detected
in 15 (35.7%), 30 (71.4%), and 12 (28.6%) of the adenomas, respectively.
Promoter hypermethylation of the p15
INK4b
gene coincided with p16
INK4a

alteration and/or RB1 methylation, whereas p16
INK4a
and

RB1 methylations
tended to be mutually exclusive (I 0.019). Thus, the vast majority of the
adenomas (38/42, 90.5%) displayed alterations of the RB1 pathway. None
of the clinicopathological features including the proliferation cell index was
signicantly correlated with any particular methylation status. Our results
suggest that inactivation of the RB1 pathway may play a causal role in
pituitary tumorigenesis, with hypermethylation of the p16
INK4a
gene being
the most common deregulation, and further provide evidence that RB1
and p16
INK4a
methylations tend to be mutually exclusive but occasionally
coincide with p15
INK4b
methylation.
339. COMPREHENSIVE GENE EXPRESSION ANALYSIS
OF HUMAN MALIGNANT GLIOMA USING CDNA
MICROARRAY FOR THERAPEUTIC STRATEGY
H. Takahashi,
1
J. Kouno,
1
and A. Teramoto
2
;
1
Neurosurgery, Nippon
Medical School Dai-ni Hospital, Kanagawa;
2
Neurosurgery, Nippon
Medical School Hospital, Tokyo; Japan
The treatment of malignant brain tumors, especially malignant glio-
mas, is still extremely difcult, and there are many indistinct points in the
molecular mechanisms participating in growth and development of malig-
nant brain tumors. Thus we tried to extract related genes by comparing a
gene expression prole of malignant glioma according to malignancy grade
and to apply such results to a clinical treatment. We extracted mRNA from
22 surgical specimens, comprising 8 diffuse astrocytomas (grade II), 6
anaplastic astrocytomas (grade III), 8 glioblastomas (grade IV), by a con-
ventional method, and the microarray analysis of complementary DNA
(cDNA) using commercial human brain total RNA (Clontech company)
as control was done. The interferon (IFN) effect prediction tip on which
775 genes were selected (MBC Company) was used for analysis of human
malignant glioma as the basis for building a therapeutic strategy. Manifest
changes were recognized by 32 genes when having compared grade IV with
grade II, and signicant variations were recognized by 12 out of 32 genes.
There was restraint tendency of gene expression level in grade IV generally,
and in particular expression of interferon reference genes were restrained.
We were able to identify the gene cluster which varied with malignancy
grade by analyzing a gene expression prole with cDNA microarray. This
study revealed that the building of a therapeutic strategy against grade IV
astrocytomas, which are very difcult to treat, is enabled on the basis of
this gene information.
340. COMBINED IMMUNOCHEMOTHERAPY WITH
REDUCED DOSE WHOLE BRAIN RADIOTHERAPY (WBRT)
FOR NEWLY DIAGNOSED PATIENTS (PTS) WITH PRIMARY
CNS LYMPHOMA (PCNSL)
G. Shah,
1
R. Lai,
2
J. Yahalom,
1
D. Correa,
1
L. DeAngelis,
1
J. Raizer,
3

D. Schiff,
4
R. LaRocco,
5
B. Grant,
6
and L. Abrey
1
;
1
Memorial-Sloan
Kettering Cancer Center, New York, New York, USA;
2
McMaster
University, Hamilton, Ontario, Canada;
3
Neurology, Northwestern
University, Chicago, Illinois, USA;
4
Neurology, University of Virginia
Medical Center, Charlottesville, Virginia, USA;
5
Kentuckiana Cancer
Institute PLLC, Louisville, Kentucky, USA;
6
University of Vermont,
Burlington, Vermont, USA
High-dose methotrexate (MTX)-based chemotherapy in combina-
tion with WBRT for pts with newly diagnosed PCNSL has been shown
to improve disease control and survival. However, disease recurrence is
common, and treatment-related neurotoxicity is an increasingly recog-
nized complication. The addition of rituximab has improved survival and
disease control in systemic non-Hodgkins lymphoma. In a phase 2 trial
we added rituximab to MTX, procarbazine, and vincristine (R-MPV) and
decreased the dose of WBRT in pts who achieved a complete response (CR)
after chemotherapy to diminish the risk of treatment-related neurotoxicity.
Twenty-seven (12 men; median age 57, range 3271; median KPS 80%) of
a planned 30 pts were enrolled from August 2002 to October 2004. Three
patients deemed ineligible for the study were not treated. Each pt received
rituximab 500 mg/m
2
on day 1 and MTX 3.5 g/ m
2
with vincristine 1.4
mg/m
2
on day 2. Procarbazine 100 mg/m
2
a day was given for seven days
during odd-numbered cycles. Pts achieving a CR after 5 cycles received
dose-reduced WBRT (2340 cGy), while pts with less than a CR received 2
more cycles, after which they received standard WBRT if a CR was still not
achieved. All pts received two cycles of Ara-C 3 g/m
2
after WBRT. Sixteen
pts were followed with prospective neuropsychological evaluations. CSF
serum levels of rituximab were assayed in pts with an Ommaya reservoir.
Fifteen of the 23 treated pts have been assessed for response after R-MPV
(ve pts are still on study, two pts were taken off study for toxicity including
grade 3 nephrotoxicity and death from neutropenic sepsis in one pt each,
and data on one pt is still being gathered). Eleven of the fteen (73%) had
a CR, two (13%) a partial response (PR) for an overall response rate of
93%, and two (13%) progressed. The median number of cycles of R-MPV
received was ve, with four patients needing seven cycles to achieve a CR.
The eleven pts with a CR received 2340 cGy of WBRT, while one pt with
a PR received 4500 cGy and one received 3640 cGy. The most common
grade 3 or 4 toxicities were granulocytopenia (34%), hyponatremia (24%),
and hyperglycemia (19%) followed by hepatotoxicity, thrombocytopenia,
coagulopathy, hypophosphatemia, hypokalemia, and anemia (10%15%
each). No grade 3 or greater neurotoxicity has been reported to date. The
safety and efcacy of R-MPV is comparable to MPV. Further investigation
of this regimen in PCNSL is warranted.
341. VACCINATION OF RECURRENT GLIOMA PATIENTS
WITH TUMOR LYSATE-PULSED DENDRITIC CELLS ELICITS
IMMUNE RESPONSES: RESULTS OF A CLINICAL PHASE 1/2
TRIAL
J. Homa,
1
R. Yamanaka,
1
T. Abe,
2
M. Narita,
2
M. Takahashi,
2
and
R. Tanaka
1
;
1
Department of Neurosurgery, Brain Research Institute,
Niigata University;
2
Department of Hematology, Niigata University
School of Medicine, Niigata University, Niigata; Japan
The objective of this study was to investigate the safety and immuno-
logical responses of dendritic cells therapy pulsed with autologous tumor
lysate in patients with malignant glioma. Twenty-two patients with malig-
nant glioma (16 and 6 patients with grade 4 and grade 3 gliomas, respec-
tively) entered in the phase 1/2 clinical study of dendritic cell vaccination.
All patients were recurrent malignant glioma patients who were resistant to
the standard maximum therapy. Dendritic cells were seven days maturation
in GM-CSF (1000 IU/ml) and IL-4 (500 IU/ml), RPMI-1640 supplemented
with 1% autologous serum, pulsed with autologous tumor cell lysate and
activated with OK 432 (0.1KE/ml). The mean numbers of vaccinations of
tumor lysate-pulsed dendritic cells were 6.2 times intradermally close to a
cervical lymph node and 4.7 times intratumorally via an Ommaya reservoir.
Clinical responses were 1 partial response, 2 minor responses, and 8 cases
of no change evaluated by radiological ndings. Dendritic cell vaccination
elicited T-cell-mediated antitumor activity, as evaluated by ELISPOT assay
after vaccination in 6 of 14 tested patients. This protocol was generally
well tolerated. This study demonstrated the safety and antitumor effects
of autologous tumor lysate-pulsed dendritic cell therapy for patients with
malignant glioma.
342. BEVACIZUMAB AND CPT-11 IN THE TREATMENT OF
RELAPSED MALIGNANT GLIOMA
V. Stark-Vance; Neurosciences, Presbyterian Hospital, Dallas, Texas,
USA
Bevacizumab (Avastin) is a monoclonal antibody that binds to and
inhibits the biologic activity of human vascular endothelial growth fac-
tor (VEGF). CPT-11 is a semisynthetic camptothecin that binds to and
inhibits DNA topoisomerase I, an enzyme necessary for DNA replication.
This combination of agents has shown clinical benet in a clinical trial
of metastatic colorectal cancer, while bevacizumab alone was associated
with inferior survival in the same population. In phase 2 clinical trials of
CPT-11 in patients with recurrent glioma, 4/59 pts demonstrated tumor
regression. Some malignant gliomas overexpress VEGF, suggesting that
bevacizumab may have clinical activity in this population, possibly increas-
ing the response rate over CPT-11 alone. Between March and December
2004, 21 patients with malignant glioma treated with this combination
(BC) were evaluated for treatment response and toxicity. A 6-week cycle
consisted of bevacizumab, 5 mg/kg, every other week 2, and CPT-11,
125 mg/m
2
every week 4, followed by a 2-week rest. MRI scans were
obtained after each cycle of treatment. Bevacizumab was not dose-reduced,
but CPT-11 doses were reduced for grade 3 or 4 myelosuppression. Of the
21 pts, 11 were GBM and 10 were other high-grade gliomas; median age
was 42 (3073) and median number of prior treatments was 3 (210). Tox-
icities included neutropenia, diarrhea, epistaxis, emesis, and asthenia. One
pt died of an intracranial hemorrhage and one pt died of complications of
gastrointestinal perforation; both are previously reported toxicities of beva-
cizumab. Four additional pts died of non-treatment-related complications.
Of the 21 pts who could be evaluated for response, there were 1 CR, 8 PRs,
and 11 SD. Thirteen pts remain on treatment, 5 after more than 4 cycles.
In all pts, radiographic responses were accompanied by reduction in both
peritumoral edema and contrast enhancement; most patients who did not
meet the criteria for partial response did show clinical and/or radiographic
improvement. These early results suggest an improvement in response rate
and duration of response over treatment with CPT-11 alone and a possible
role for bevacizumab in primary therapy for high-grade glioma.
343. THE FAS/FAS LIGAND PATHWAY FAILS TO INDUCE
APOPTOSIS IN EXPERIMENTAL GLIOMA CELLS AND
COULD BE RESPONSIBLE FOR THE APOPTOSIS OF T- CELL
SURROUNDING MALIGNANT GLIOMAS IN VIVO
F. Legnani,
1
G. Pradilla,
2
Q. Thai,
2
B. Tyler,
2
G. Zamora,
2
S. Gaini,
3
F.
DiMeco,
1
H. Brem,
2
and A. Olivi
2
;
1
Neurosurgery, Istituto Neurologico
C. Besta, Milan, Italy;
2
Neurosurgery, Johns Hopkins University,
Baltimore, Maryland, USA;
3
Neurosurgery, Universit degli Studi di
Milano, Milan, Italy
Human and experimental gliomas have been shown to express the
Fas-receptor (FasR). Activation of this receptor by direct agonists could
trigger caspase-3/8-mediated apoptosis. FasR-agonist-mediated apoptosis
of tumor invading lymphocytes has also been demonstrated. Therefore,
the role of Fas-ligand (Fas-L) as an anti-tumor pathway remains contro-
versial. In this study we tested the ability of recombinant-FasL and of a
FasR-agonist-antibody (Ab) to induce apoptosis in 9L cells, and we explain
the tumor immune-evasion in the 9L-gliosarcoma model. FasR expression
was determined in 9L, F98, U257, and U373 cells by Western blot. Cyto-
toxicity of recombinant-FasL and FasR-agonist-Ab was evaluated in vitro
against 9L. Cells were treated with 50, 100, 250, and 500 ng/ml of FasL
and FasR-Agonist-Ab. Percentage of cell viability was established by using
the MTT assay. Statistical analysis was done using the Student t test (P
0.05 was considered signicant). For the in vivo studies, 33 Fischer 344
rats were intracranially implanted with 9L and perfused/xed on day 10.
Tumor samples were processed for immunohistochemical staining and con-
focal microscopy analysis. Monoclonal antibodies were used against CD3
(Mouse monoclonal, Serotec), Fas-L (Rabbit polyclonal, Santa Cruz Bio-
technology), GFAP (Mouse monoclonal, NeoMarkers; Rabbit polyclonal,
DAKO), and caspase-3 (Rabbit polyclonal) to identify lymphocytes, Fas-L,
astrocytes, and apoptotic lymphocytes. A robust band at 48 kDa conrmed
strong FasR expression in all cell lines tested. Treatment with recombinant-
FasL at 50 ng/ml and 500 ng/ml decreased cell viability to 98% 0.03%
and 95% 0.03%, respectively. Similarly, treatment with FasR-agonist-Ab
at 50 ng/ml and 500 ng/ml decreased cell viability to 93% 5% and 96%
1%, respectively. Percentage of growth was statistically insignicant
for the tested concentrations. Compared to controls, animals intracrani-
ally challenged with 9L exhibited signicantly higher expression of FasL,
caspase 3, and CD3 cells. Apoptotic CD3 cells were identied by co-
localization of CD3 and caspase 3. CD3 cells were identied peri- and
intra-tumorally by distinctive clustering patterns in the center of the tumor.
Although FasR was strongly expressed in all cell lines tested, the treatment
with FasL and FasR-agonist-Ab failed to induce apoptosis. Under these
experimental conditions, both FasR-agonist-Ab and recombinant-FasL are
ineffective in treating brain tumors. Rats intracranially implanted with 9L
gliosarcoma exhibit marked upregulation of Fas-ligand in GFAP tumor
cells. The presence of apoptotic CD3 cells suggests that a Fas-ligand-
mediated immune-evasion mechanism is responsible for T-lymphocyte
apoptosis in the 9L gliosarcoma model.
344. GPNMB: A NEW TARGET FOR HUMAN HIGH- GRADE
GLIOMAS (HGL) IMMUNOTHERAPY
C. Kuan,
1
K. Wakiya,
1
J.M. Dowell,
2
J. Herndon,
2
G.J. Riggins,
3
M.
Graner,
1
C.J. Wikstrand,
1
and D.D. Bigner
1
;
1
Pathology and
2
Cancer
Center Biostatistics, Duke University Medical Center, Durham, North
Carolina;
3
Neurosurgery, Johns Hopkins University, Baltimore,
Maryland; USA
Targeting neoplastic HGL cells for treatment with monoclonal antibody
(MAb) constructs requires markers expressed on the cell surface of gliomas
but not normal brain. We here identied promising new HGL tumor tar-
gets, human transmembrane glycoprotein nmb (GPNMB
wt)
, and a splice
variant form (GPNMB
sv
) leading to an in-frame insertion of 12 amino acids
in the extracellular domain (ecd). We have performed genetic and immuno-
histochemical (IHC) evaluation of human gliomas to determine incidence,
distribution, and pattern of localization of GPNMB antigens in brain
tumors as well as survival analyses. We have generated anti-GPNMB anti-
bodies by immunizing rabbits and mice with plasmid DNA coding the ecd
of GPNMB, followed by boost with corresponding recombinant proteins.
To determine the GPNMB mRNA transcript and protein expression levels
in glioma samples, we have performed quantitative RT-PCR (49 cases) and
IHC (140 cases) in HGL patient cases and glioma GPNMB transfected cell
lines. We also characterized ve monoclonal antibodies using Western blot-
ting, BIAcore, Scatchard, and FACs analyses. Survival analyses were also
conducted based upon IHC information from 80 newly diagnosed patients
(63 glioblastoma multiforme [GBM] and 17 anaplastic astrocytomas [AA])
and the RNA expression data from 29 newly diagnosed patients (26 GBM
and 3 AA). The real-time PCR results from 49 HGL biopsies indicated
31/36 (86%) GBM, 3/6 (50%) AA, and 3/7 (43%) anaplastic oligoden-
drogliomas (AO) were positive for gpnmb RNA transcripts (gpnmb
wt
plus
gpnmb
sv
). gpnmb
sv
was detected in 16/36 (44%) GBM and 4/6 (67%) AA.
In normal brain samples, little or no expression was noted for GPNMB
wt

and GPNMB
sv
mRNA. We have obtained and characterized anti-GPNMB
polyclonal rabbit antiserum #2640 as well as two IgG
1
MAbs, G11 and
A3, and three IgG
2b
MAbs, U2, U4, and U5. The binding afnity constant
K
A
of MAbs to GPNMB was 2.7 to 96 10
7
M
-1
and 7.6 to 47 10
7

M
-1
by BIAcore and Scatchard analysis, respectively. A larger HGL study
(n 140) revealed detectable GPNMB by IHC with antiserum #2640 and
MAb G11 in a membranous and cytoplasmic pattern, with occasional focal
perivascular reactivity in 21/32 (66%) AA and 75/108 (70%) GBM. Quan-
titative ow cytometric analysis of 11 GPNMB positive GBM fresh biopsy
specimens revealed GPNMB cell surface molecular density at 1.1 to 7.8
10
4
molecules/cell, levels sufcient for MAb targeting. The survival analysis
demonstrated newly diagnosed AA/GBM patients over the age of 45 have
a higher risk of death (hazard ratio of 2.5). Signicantly, in this popula-
tion (AA/GBM or GBM alone) univariate analyses show that patients with
relative mRNA transcript levels greater than 3-fold over normal brain also
have a higher risk of death (hazard ratio of 5 to 7). Increased mRNA levels
correlated with higher survival risk and elevated protein expression in HGL
biopsy samples, combined with the detection of surface membrane proteins
in glioma cells, indicates that GPNMB is a valuable tumor-associated anti-
gen in immunotherapeutic approaches for malignant gliomas.
345. IMMUNIZATION WITH AUTOLOGOUS GLIOMA
CELLS TRANSFECTED WITH IFN- G GENE SIGNIFICANTLY
PROLONGS SURVIVAL IN GBM-PATIENTS OLDER THAN 50
YEARS
L. Salford,
1,2
E. Ask,
3
P. Siesj,
1,2
G. Skagerberg,
1,2
C. Baurus-Koch,
2,4

C. Blennow,
1,2
A. Darabi,
1,2
C. Elfgren,
5
E. Englund,
6
S. Janelidze,
1,2

E.-M. Larsson,
7
. Lilja,
1
B.R.R. Persson,
2,4
A. Rydelius,
1,3
S.
Strmblad,
1,2
E. Visse,
1,2
and B. Widegren
7
;
1
Department of
Neurosurgery,
2
The Rausing Laboratory, and Departments of
3
Neurology;
4
Medical Radiation Physics,
5
Neuropsychology,
6
Neuropathology,
7
Neuroradiology, and
8
Tumor Immunology, Lund
University, Lund, Sweden
Based upon earlier experimental work by our group, we have com-
pleted the rst part of a human immuno-gene therapy study of patients
who undergo an operation for a glioblastoma multiforme. The goal is to
ascertain whether immunization with autologous tumor cells expressing
gene sequences for human IFN-g is safe for the patients, gives rise to an
immunological response, and adds any benecial effect to conventional
therapy. This report is based upon the completed treatment of our rst nine
patients. Patients aged 50 to 69 years, in which at least 80% of the GBM
is resected during neurosurgery, can be included in the study. The tumor
cells are cultured in a special Clinical Transduction Laboratory. When at
least 40 million cells, karyotyped as malignant, are collected, the immu-
nizations are given 3-weekly, repeated at least 4 and at most 10 times. The
immunization takes place in the dermis of the upper arm. Seven days after
each immunization, a skin biopsy is taken from the centre of one of the
injection sites. The composition of the cellular inltration in the skin is
studied by markers for T lymphocytes (CD3); helper cells, subset of T cells
(CD4); cytolytic T-cells, subset of T cells (CD8); natural killer cells (CD16);
and B lymphocytes, B cells (CD20). Also the expression of cytokines for
functional T cell subsets is studied: IL-2, IL-4, IL-10, IL-12, IL-18, TNF-a,
TNF-, IFN-g and TGF-b 1, 2 and 3. Peripheral blood is sampled both before
and after operation and also after each immunization event. Co-culture
of this blood with tumor cells from the patient allows for a selection of T-
cells that can recognize tumor-specic antigens. The patients are followed
regularly with neurological, neuropsychological, and MRI examinations.
Results from the rst human treatments show that the method is safe for
the patients and that it gives rise to positive DTH reactions and an increase
of inltrative CD8 and CD4 T-cells at the site of immunization. Seven
patients received 10 immunizations, and 1 had eight and 1 six immuniza-
tions. The material is small, but out of 9 patients, 3 survived for 19.5, 22,
and 26.5 months and one is still alive after 23 months with a minor tumor
burden. The mean survival time of these 9 patients (mean age 62 years) is
16.2 months to be compared to the 10.4-month survival (P 0.03, Mann-
Whitney one-sided test) of the 13 patients (mean age 60 years) included in
the study but where the cells did not grow sufciently well in the cultures
to make immunization possible.
346. PRE- CLINICAL EVALUATION OF D2C7, A
MONOCLONAL ANTIBODY REACTIVE FOR BOTH
THE WILD TYPE AND VARIANT III MUTANT
EPIDERMAL GROWTH FACTOR RECEPTOR, FOR
RADIOIMMUNOTHERAPY OF MALIGNANT GLIOMAS
A. Boskovitz,
1
C. Pegram,
1
K. Peixoto,
2
M.R. Zalutsky,
2
and D.D.
Bigner
1
; Departments of
1
Pathology and
2
Radiology, Duke University
Medical Center, Durham North Carolina, USA
The epidermal growth factor receptor (EGFR) is expressed by normal
epithelial cells in most tissues but overexpressed in 71%94% and 60%
90% of anaplastic astrocytomas and glioblastomas (GBM), respectively.
In addition, 58%61% of GBM also express the EGFR variant III mutant
(EGFRvIII), which is not found on normal tissues. Monoclonal antibod-
ies (MAbs) targeting either the wild-type EGFR (wt) or EGFRvIII have
been developed, and one of them (MAb 425) has been introduced in radio-
immunotherapeutic trials for patients with malignant gliomas. Although
EGFRvIII is a preferred target because of its higher tumor specicity, some
glioma cells may express only wt. Herein, we evaluated the biologic prop-
erties of D2C7, a novel MAb specic for both wt and EGFRvIII, with the
hypothesis that it could serve as a more generally applicable radionuclide
carrier for the targeted radiotherapy of patients with malignant gliomas.
D2C7 was generated by using hybridomas reacting with the extracellular
domain (ecd) of wt or EGFRvIII. The afnity for wt and EGFRvIII was
determined by surface plasmon resonance. Paired-label experiments com-
pared the tissue distribution of
125
I-labeled D2C7 and
131
I-labeled anti-wt
MAb EGFR1 (Exp. 1),
131
I-labeled control MAb P588 (Exp. 2 and 4), or
131
I-labeled anti-EGFRvIII MAb L8A4 (Exp. 3) in a subcutaneous xeno-
graft model expressing selectively wt (U87-wt; Exp. 1 and 2) or EGFRvIII
(NR6M; Exp. 3 and 4) in athymic mice. The binding afnity (K
d
) of D2C7
against the wt and EGFRvIII ecd was 1.93 10
-10
M
1
and 2.8 10
-10
M
1

respectively. In vivo, wt-expressing tumor uptake of
125
I-labeled D2C7 was
30.8 3.0 (Exp. 1) and 34.3 9.6 (Exp. 2) percent injected dose (ID)/g
at 24 h and declined thereafter. The tumor localization index of D2C7
versus EGFR1 and P588 was 3.4 0.9 and 3.3 0.4, respectively, at 24
h and increased thereafter.
125
I-labeled D2C7 uptake at 24 h in EGFRvIII-
expressing tumors was 37.0 4.2 (Exp. 3) and 41.7 17.6 (Exp. 4) percent
ID/g and increased to peak at 52.5 14.0 and 81.8 8.6 percent ID/g,
respectively, 72 h after injection. The tumor localization index of D2C7
versus L8A4, an EGFRvIII specic MAb, and P588 was 2.0 0.1 and 2.9
1.0 at 24 h and 3.0 0.9 and 5.8 1.0 at 72 h, respectively. In vitro,
D2C7 demonstrated strong afnity and delayed off-rate for both the wt
and EGFRvIII molecules. In vivo, tumor localization of D2C7 in tumors
expressing wt or EGFRvIII was signicantly higher than that of established
specic MAbs. In correlation with the in vitro data, uptake of D2C7 by
EGFRvIII-expressing tumors was higher compared to that of wt-expressing
lesions. Further investigations of the D2C7 MAb are in progress to assess
its therapeutic potential as a radiolabeled targeting agent for EGFRvIII-
expressing malignant gliomas. Because of the presence of wt on some nor-
mal tissues, compartmental administration is preferred in order to reduce
potential targeting of normal tissues.
347. EXTERNALLY APPLIED MONOCYTES INFILTRATE
GLIOMA SPHEROIDS AND RAT GLIOMAS
H. Strik,
1
P. Erdlenbruch,
2
P. Hlper,
2
J. Maier,
1
A. Kowalewski,
1

B. Hemmerlein,
3
R. Gold,
4
and M. Bahr
1
;
1
Neurology,
2
Pediatrics,
3
Pathology,
4
MS Research, Georg August University, Gttingen,
Germany
One prominent feature of human gliomas is the lack of cellular anti-
tumor immune response despite extensive inltration with mononuclear
cells. We established a model of monocytic invasion into glioma cell spher-
oids in vitro and showed that monocytes injected into the peripheral blood
inltrate rat gliomas in vivo. Monocytes were isolated from human blood
and added to precultured spheroids of U118 or A172 glioma cells. After
three days, the spheroids were collected, xed and examined immunohis-
tochemically. Peritoneal macrophages were stained with the uorescent
marker PKH2, collected after 24 h, and injected into RG2-glioma-bearing
Fischer rats intravenously or into the carotid artery. After 24 h, the rats
were sacriced and frozen sections of the brains searched for uorescent
macrophages. Spheroids were inltrated regularly by monocytes in vitro.
The monocytes expressed MRP8, MAC387 (MRP14), and 25F9. In the
rat glioma model, accumulation of the marked monocytes was observed
24 h after injection into the peripheral blood. The experimental models
presented here allow us to analyze interactions between monocytes and
glioma cells both in vitro and in vivo. A better understanding of these inter-
actions may help to overcome the inactivation of the monocytic anti-tumor
response by glioma cells.
348. EXPRESSION OF 8 TUMOR ANTIGENS BY
QUANTITATIVE-POLYMERASE CHAIN REACTION IN A
SERIES OF 50 GLIOBLASTOMAS
V. Quillien,
1
B. Collet,
1
S. Saikali,
2
T. Avril,
3
and Y. Guegan
2
;
1
Biology,
Anti-cancer Center Eugene Marquis, Rennes;
2
Neuro-Oncology
University Hospital, Rennes;
3
UPRES EA 3889, Medical School, Rennes;
France
The prognosis of glioblastoma (GBM) remains particularly poor and
needs development of new therapeutic strategies. Recent pilot studies have
shown that immunotherapy could be of interest in GBM. Currently, pro-
tocols use whole autologous tumor as source of antigenic peptides (intact
tumor cells, tumor lysates or peptides eluted from autologous tumors)
because no denitive glioma-specic antigens have been identied to date.
This approach has drawbacks among which are the potential of initiating an
auto-immune response against brain normal antigens, the delay required to
have enough tumor cells after a possible step of culture, and the difculties
to analyze specic anti-tumor immune responses. In a series of 50 GBMs,
we have tested mRNA expression of 8 tumor-associated antigens, ALK
(anaplastic lymphoma kinase), IL13R2, gp100, Galt3, tyrosinase, TRP-2,
NA-17A, and MAGE-A3, by Q-PCR (quantitative-polymerase chain reac-
tion). All these antigens have previously been identied in brain tumor,
often in small series of various histologies. In addition, they all contain
HLA-A2-restricted CD8 T-cell epitopes in their sequence. All GBMs were
classied according to the WHO. Samples were stored at 80C until RNA
isolation. Quantitative gene expression was performed on a Taq Man ABI
PRISM 7000 using predesigned probe and primer sets (Applied Biosystems).
GAPDH was used as internal housekeeping gene. RNA sample expression
was normalized to the expression obtained from an RNA pool of 4 normal
brain tissues. Ratios (sample RNA level/normal brain RNA level) above 2
were considered as positive. Frequencies of expression were observed as fol-
lows: ALK, 8%; NA-17A, 8%; MAGE-A3, 12%; TRP-2, 38%; IL13R2,
60%, and gp100, 76%. No expression was found for tyrosinase and Galt3.
Gp100, IL13R2, and TRP-2 were simultaneously expressed in 24% of
the GBMs; 38% of GBMs expressed two of them, and 84% expressed one
of them. Mean ratios (minimum ratio maximum ratio) were 7 (240) for
gp100, 7 (29) for IL13R2, and 16 (2155) for TRP-2. More than 70%
of the samples had an expression ratio less than 10. Gp100, IL13R2, and
TRP-2 may represent useful targets for specic immunotherapy. We are
now testing their capacities to elicit a specic cytotoxic immune response in
vitro in peripheral blood lymphocytes from patients with GBM.
349. THE STUDY OF INDUCING ANTI- GLIOMA ACTIVITY
IN VITRO WITH HUMAN AUTOLOGOUS DENDRITIC
CELL-BASED FUSION CELLS
Z. Chen and H. Shi; Neurosurgery/Neuro-oncology, Cancer Center,
Guangzhou, China
As effective antigen-presenting cells, dendritic cell (DC)-based hybrid
vaccine has shown much promise in cancer therapy. But there have been
few approaches in human glioma treatment, and it is an area of research
to be addressed in our country. In this study, DC-glioma fusion cells (FC)
were prepared by the fusion of human glioma cells and the DC derived
from peripheral blood mononuclear cells of the same patients. In vitro anti-
glioma activity induced by FC was studied. The FC were prepared in the
presence of polyethylene glycol, the proliferation capacity of a patients T
cells in vitro stimulated by FC was evaluated by autologous mixed leuko-
cyte reaction, and the cytotoxicity against autologous glioma cells by the
patients T cells primed with FC was also assessed by lactate dehydrogenase
(LDH) assay. The FC-preparation method was constructed. The patients T
cells primed with irradiated (1.5 Gy) FC induced a more remarkable glioma
killing than the T cells primed with DC or autologous glioma cells (P
0.01), and the cytolytic effects were up from 28% to 90% as the effector-
target ratio increased. But irradiated FC primed T cells only induced about
10% MCF7 lysis, which was signicantly lower than glioma killing (P
0.01). When stimulated, the FC primed T cells showed remarkably high
proliferation compared with DC or autologous glioma cells primed T cells
(P 0.05), and those primed T cells stimulated with autologous DC loaded
with glioma lysate showed signicantly enhanced proliferation compared
with those stimulated with autologous glioma cells or DC alone (P 0.01).
The irradiated (1.5 Gy) FC prepared in this protocol could effectively prime
a patients T cells, and the primed T cells could be proliferated in vitro as
stimulated with autologous DC loaded with glioma lysate. The primed T
cells showed a specic tumor cytolysis, and the killing activities were up as
the effector-target ratio increased. These studies may have implications for
the clinical trials on human gliomas.
350. CULTURING OF HUMAN TUMOR CELLS FOR USE IN
IMMUNE GENE TUMOR THERAPY
B. Widegren,
1,4
C. Baureus-Koch,
5
A. Rydelius,
2
G. Nyberg,
2
S. Jrnum,
1

A. Darabi,
2
P. Siesj,
2
E. Visse,
2
S. Strmblad,
2
C. Blennow,
2
G.
Skagerberg,
2
X. Fan,
1
N. Mandahl,
3
H.-O. Sjgren,
1
B.R.R. Persson,
5

and L.G. Salford
2
;
1
Tumor Immunology,
2
Division of Neurosurgery, The
Rausing Laboratory, and Departments of
3
Clinical Genetics,
4
Molecular
Genetics, and
5
Medical Radiation Physics, University Hospital, Lund,
Sweden
The study concerns the culturing of tumor cells from brain tumor speci-
mens to provide a source of tumor antigens for immunization purposes. It is
important here to be able to discriminate normal cells from malignant cells
in glioma cell cultures effectively. We outline a protocol for enhancing pri-
mary tumor cell growth in vitro from tissue obtained during surgery from
glioblastoma multiforme. Cancer immune therapy utilizing autologous
human tumor cells, genetically modied to enhance their immunogenic-
ity, is a method currently under development and successful clinical results
have been published. Ongoing and planned clinical trials involve immuni-
zation with irradiated tumor cells as a method of choice, especially when
the actual tumor antigens have not yet been identied. For immunotherapy
using autologous human tumor cells to be successful, several million tumor
cells are needed. The time elapsing between tumor biopsy and treatment
should be as short as possible. Using tumor material directly will involve
normal cells that increase the risk of autoimmunity. For many tumors,
including human gliomas, however, the establishment of in vitro growth is
not always possible. We have investigated the in vitro growth potential of
human glioma biopsies from more than 30 patients. This involved testing
different matrices (laminin, collagen, and bronectin), supplemented by
different growth factors (either platelet-derived growth factor [PDGF] or
epidermal growth factor [EGF]), and using different batches of sera. The
effect of steroids was also studied. To investigate the ratio of normal to
malignant cells in the tissue cultures from the tumor biopsies, chromosomal
analysis of the glioma cell cultures was performed. Since for most tumors,
no tumor-protein marker exists that is able to adequately discriminate a
glioma cell from a normal cell, high-resolution chromosomal analysis is the
safer method. Establishing adherent human glioma cell cultures from sur-
gical biopsies was successful in about 75% of the cases. Enhanced growth
could sometimes be observed when cells were supplemented with EGF or
PDGF, whereas the growing of cells in asks coated with matrix proteins
only rarely produced substantial improvements in growth. High-resolution
chromosomal analysis of the cultures demonstrated the presence of normal
cells in amounts ranging from 0% to almost 100%. The general tendency
is for malignant cells to be present in cultures in large numbers initially but
rather for normal cells to outnumber the malignant cells quickly. Later on,
the normal cells cease to grow, and the tumor cells become dominant. The
time elapsing between biopsy and development of a cell culture in which
the majority of the cells are malignant varies from a few weeks to more than
six months. The low frequency of tumor cells in cultures at certain stages
makes the cultures unsuitable at such points for immunogene therapy with
use of autologous tumor cells. We also demonstrate efcient transfection
results with the use of adenovirus expressing human interferon gamma and
green uorescent protein.
351. TRAIL (APO2L) AND TRAIL-RECEPTOR EXPRESSION
IN RELATION TO SURVIVAL OF PATIENTS WITH
J.M.A. Kuijlen,
1
J.J.A. Mooij,
1
H. Hollema,
2
E.W. Hoving,
1
W.T.A. van
der Graaf,
3
and W.F.A. den Dunnen
2
;
1
Neurosurgery,
2
Pathology and
Laboratory Medicine and
3
Internal Medicine, Groningen University
Hospital, Groningen, The Netherlands
In order to improve survival of patients with a GBM, new therapeutic
strategies must be developed. The use of a death-inducing ligand such as
TRAIL (TNF-related apoptosis-inducing ligand) seems a promising inno-
vative therapy. The aim of this study was to quantify the expression of
the death regulating receptors TRAIL-R1 and TRAIL-R2, and the TRAIL
ligand on primary GBM tumor specimens and to correlate this expression
with survival. Sixty-two tumor specimens were taken from patients who
had had a craniotomy with debulking of a primary GBM. Expression of
TRAIL and TRAIL receptors was assessed by immunohistochemistry, both
quantitatively (% of positive tumor cells) and semi-quantitatively (staining
intensity) within both perinecrotic and intermediate tumor zones. RT-PCR
of GBM tumor tissue and controls (other astrocytic tumors and normal
brain) was performed to show expression of TRAIL receptor mRNA.
Immunohistochemistry showed a slight diffuse intracytoplasmic and a
stronger membranous staining for TRAIL and TRAIL receptors in tumor
cells. Semi-quantitative expression of TRAIL showed a signicantly higher
expression of TRAIL in the perinecrotic area than in the intermediate zone
of the tumor (P 0.0001). A mean of 13% TRAIL positive tumor cells
was found. A positive correlation was found between TRAIL expression
(semi-quantitative) and survival (P 0.008). Mean tumor cell positivity
for TRAIL-R1 was 19%. A positive correlation was found between sur-
vival and the percent of TRAIL-R1 positive tumor cells (P 0.049). Mean
TRAIL-R2 expression was 27%. TRAIL-R2 expression was signicantly
higher expressed than TRAIL-R1 (P 0.005). A positive correlation was
found between TRAIL-R1 and TRAIL-R2 expression (P 0.002). RT-
PCR analysis detected a negative relation between the amount of TRAIL-R1
mRNA and the WHO grade of astrocytic tumors (P 0.005). Immunohis-
tochemistry and RT-PCR show a positive correlation between TRAIL-R1
expression and survival, and therefore TRAIL-R1 expression seems to be
a prognostic indicator. TRAIL-R2 is signicantly more expressed within
tumor tissue than TRAIL-R1. Therefore TRAIL-R2 is of more importance
as a target for future TRAIL therapy than TRAIL-R1. However, the way
the death-inducing signal through the TRAIL-R2 receptor is regulated dif-
fers from TRAIL-R1 signaling. This has to be taken into account when
developing new therapeutic strategies using TRAIL.
352. BCL-2 FAMILY MEMBERS, NFKAPPAB, AND
DESFEROXAMINE PROTECT T- CELL FROM
GBM-MEDIATED APOPTOSIS
A. Chahlavi,
1
M. Thornton,
2
C. Tannenbaum,
2
P. Rayman,
2

M. Vogelbaum,
1
G. Barnett,
1
and J. Finke
2
;
1
Neurosurgery and
2
Immunology, Cleveland Clinic Foundation, Cleveland, Ohio, USA
Prior studies have shown that glioblastoma multiforme (GBM) cells
and possibly tumor-derived products may be responsible for the T cell dys-
function observed in GBM patients, thus impairing T-cell based immu-
notherapy. This notion is supported by the observation that GBM tumor
cells, when co-cultured with T cells, can induce apoptosis of the lympho-
cytes. Established GBM lines are known to express gangliosides as well as
death-inducing ligands such as CD70 that can induce apoptosis in T cells.
We wanted to determine whether overexpression of Bcl-2 family members,
Bcl-2 and B-1, and RelA, a subunit of NFkappaB, will provide protection
to T cells from GBM-induced apoptosis. Also, we wanted to assess if cyclo-
sporine A, bongkrekic acid (BA), and desferoxamine, which protect against
mitochondrial damage, can protect T lymphocytes from cell death induced
following co-culture with GBM lines or GBM gangliosides. The Jurkat T
cell line was transfected with plasmids expressing the gene for Bcl-2, B-1,
or RelA, and stably transfected clones were established and conrmed by
quantitative PCR and Western and Northern blot analysis. Five GBM cell
lines were co-cultured with wild-type Jurkat cells and Jurkat cells over-
expressing the various transgenes. Jurkat cell apoptosis was measured by
trypan blue and TUNEL. The expression of Bcl-2, B-1, and RelA was
measured by quantitative PCR and Western blot in wild-type Jurkat cells
after co-culturing with GBM cell lines. Four of ve GBM cell lines induced
high apoptosis of wild-type Jurkat cells and resting or activated T-cells in
co-culture experiments. The expression levels of Bcl-2, B-1, and RelA were
subsequently reduced in the wild-type Jurkat cells following co-culture with
the apoptogenic GBM cell lines. Overexpression of Bcl-2, B-1, and RelA,
however, conferred protection by at least 60% against the four apopto-
genic GBM cell lines. As far as drug protection, all three achieved some
protection. However, desferoxamine, an iron chelator, was most effective
at blocking T-cell killing by GBM cell-lines and their puried gangliosides.
Bcl-2, B-1, and RelA overexpression is an effective means of protecting
T lymphocytes from apoptosis mediated by GBM. Furthermore, the anti-
oxidant desferoxamine may be useful in vivo as an adjunct drug in GBM
immunotherapy for promoting T-cell survival and function.
353. CYTOKINE EXPRESSION IN RG2 GLIOMAS
FOLLOWING TREATEMENT WITH CPG DNA,
INTERFERON- GAMMA, AND INTERFERON WITH LPS
J. Schartner, L. Zhang, M. Van Handel, G. Allen, M. Curet, and
B. Badie; Neurological Surgery, University of Wisconsin, Madison,
Wisconsin, USA
The efcacy of immunotherapies against malignant gliomas is likely
limited by the immunosuppressive tumor environment. Our lab has previ-
ously shown that intracranial RG2 gliomas demonstrate higher expression
of both immuno-stimulatory (IL-1b and IFN-g) and immunosuppressive
cytokines (IL-10 and TGF-b1) mRNA as compared to normal brain tis-
sue. To investigate changes in baseline cytokine expression following
immune stimulation, normal tissue and RG2 tumor tissue were analyzed
by semi-quantitative real-time PCR 24 h after injections with CpG DNA,
interferon-g (IFN-g), or IFN-g with lipopolysaccharide (IFN/LPS). CpG-
injected normal brain tissue demonstrated a 21-fold increase in IFN-g and
IL-1b and an 1160-fold increase in IL-10 mRNA as compared to sham-
treated normal brain. TGF-b1 or TGF-b2 expression did not change in
these samples. Although CpG-treated RG2 tumors also demonstrated
an increase in IL-10 (363-fold), IFN-g (48-fold) and IL-b1 (only 8-fold)
mRNA levels, they also demonstrated an increase in TGF-b2 expression
(10-fold). Treatment of normal brain or RG2 tumors with IFN-g did not
change the expression of any cytokines in normal brain, but induced a
5.6-fold increase in TGF-B2 mRNA in RG2 tumors. IFN/LPS treatment
increased the expression of IFN-g, IL-1b, and IL-10 in normal brain (13.9-,
34-, and 15-fold increases over sham-treated normal brain). However, these
effects were limited in RG2 tumors, with no change in IFN-g expression
and only a 6-fold change in IL-1B expression compared to sham-treated
tumors. Overall, these observations suggest that treatment of RG2 tumors
with classical immune activating agents may result in increased expression
of pro-inammatory cytokines, but that the concomitant upregulation of
immuno-suppressive cytokines may limit the efcacy of such treatment.
Future therapeutic strategies will need to address pervasive immunosup-
pressive environment present in these tumors.
354. IMMUNE REJECTION OF GLIOMAS IN A MURINE
MODEL
S. Jeyapalan,
1
M. Pasternack,
2
D. Knipe,
4
F.H. Hochberg,
5
and
K. Reilly
3
;
1
Neurology, Beth Israel Deaconess Medical Center, Boston,
Massachusetts;
2
Pediatrics, Massachusetts General Hospital, Boston,
Massachusetts;
3
National Institutes of Health, National Cancer Institute,
Bethesda, Maryland;
4
Microbiology, Harvard Medical School, Boston,
Massachusetts;
5
Neurology, Massachusetts General Hospital, Boston,
Massachusetts, USA
Viral vectors encoding therapeutic genes are being investigated as
novel agents for glioma therapy. These candidate therapeutic genes may
modulate the immunogenicity of viral vectors and affect vector efcacy
to inhibit tumor growth independent of their enzymatic activities. We
investigated the immunogenicity of an exogenous gene product in a murine
model using ovalbumin (OVA) to study whether an exogenous antigen
promotes immune-mediated lysis of glioma cells. OVA-expressing glioma
lines were established by transfection and cloning of KR-158 glioma cells
(H-2b); OVA expression was conrmed by immunouorescence and West-
ern blotting. The tumorigenicity of wild-type, low- and high-expressing
OVA-transfected glioma cells to induce subcutaneous tumors in syngeneic
C57Bl/6 mice was compared. Although as few as 10
6
wild-type KR-158
glioma cells generated ultimately lethal bulky ank tumors, low- and high-
expressing OVA transfectants (6 10
6
) were not tumorigenic in C57Bl/6
mice. These transfectants remained tumorigenic in nu/nu recipients and
pointed to immune rejection of OVA-transfected gliomas. The role of the
host immune response in the rejection of OVA-expressing gliomas was
explored through the use of C57Bl/6 mutants decient in specic compo-
nents of the T-cell response. Beta
2
-microglobulin null mice did not reject
OVA-expressing KR-158 glioma cells, pointing to an essential role of MHC
class Ispecic CD8 cytotoxic T cell function in glioma rejection. Surpris-
ingly, the OVA-transfectant gliomas were not tumorigenic in perforin null
recipients, suggesting that Fas-mediated lysis, and not cytotoxic granule-
mediated killing, was required for glioma rejection. The growth of OVA-
expressing KR-158 glioma cells in gld/gld congenic mice, which lack Fas
ligand on immune effector cells, conrmed the importance of Fas-mediated
tumor cell lysis. Flow cytometric analysis of cultured KR-158 demonstrated
subpopulations expressing Fas (~20%) or Fas ligand (15%). These data
demonstrate that exogenous transgenes, modeled here by ovalbumin, are
potentially immunogenic antigens, and may enhance immune-mediated
oncolysis. As new candidate therapeutic genes are investigated, their impact
on the immunogenicity of therapeutic viral vectors and their intrinsic effects
on tumor growth in vivo should be explored.
355. INCREASED NUMBER OF LEUKOCYTES SECRETING
IFN- GAMMA IN PATIENTS IMMUNIZED WITH
AUTOLOGOUS IFN- GAMMA SECRETING GLIOMA CELLS
CORRELATE WITH PROLONGED SURVIVAL
E. Visse, A. Darabi, K. Enell, S. Janelidze, M. Larsson, B. Widegren, G.
Skagerberg, P. Siesj, and L. Salford; Clinical Medicine, Neurosurgery,
The Rausing Laboratory, Lund, Sweden
In a clinical study glioma patients were immunized intradermally in the
right arm with autologous tumor cells secreting IFN-gamma. Peripheral
blood leukocytes (PBL) and immune inltration at the immunization site
were studied to monitor the patients immune response. Before and during
the 18- to 30-week immunization schedule, blood samples were collected,
and PBL were isolated and frozen. After thawing, PBL were restimulated
with tumor cells in vitro for 5 days. The restimulated cells were assayed for
lymphocyte subset distribution by ow cytometry, cytokine production by
ow cytometry, and ELIspot and proliferation by thymidine incorporation.
Immunohistochemically stained tissue sections from the immunization site
were analyzed for leukocyte inltration. Blood samples from 5 patients
were analyzed and compared with survival data, immune inltration, and
DTH reactions at the vaccination site. No correlation or discernable pat-
tern could be observed from proliferation and lymphocyte subset analyses.
Increased IFN-gamma levels measured by ELIspot were seen in 2/5 patients
with a prolonged survival. A decrease in IFN-gamma production was
observed during tumor recurrence. Detectable levels of IL-10 were observed
displaying a weak association with shorter survival. Lymphocyte cytokine
production did not correlate with lymphocyte inltration or DTH reac-
tions at the immunization site. Compared to control patients, immunized
patients had a mean prolonged survival of 170 days. No clear correlation
between immune cell inux at the immunization site and recall lymphocyte
activity or prolonged survival could be found. The mean survival time of
patients immunized with IFN-gamma secreting tumor cells is prolonged by
circa 4 months. The two patients with the longest survival time also had an
increased number of IFN-gamma producing leukocytes.
356. IL-13 RECEPTOR ALPHA 2 PEPTIDE ANALOGUES
INDUCE HIGHER LEVELS OF IL-13 RECEPTER ALPHA
2 (345-353) -SPECIFIC CTL RESPONSES IN BOTH
GLIOMA PATIENT-DERIVED CD8 CELLS AND HLA-A2
TRANSGENIC MICE
J. Eguchi,
1
M. Hatano, J. Dusak,
1
F. Lieberman,
1
W. Storkus,
2

I. Pollack,
1
and H. Okada
1
;
1
Neurosurgery;
2
Dermatology, University
of Pittsburgh, Pittsburgh, Pennsylvania, USA
Restricted and high-level expression of interleukin-13 receptor-alpha
2 (IL-13R2) in a majority of human malignant gliomas makes this pro-
tein an attractive vaccine target. We have previously described identica-
tion of IL-13R2 (345-353) peptide as an HLA-A2-restricted cytotoxic T
lymphocyte (CTL) epitope. However, as it remains unclear how efciently
peptide-based vaccines can induce specic CTLs in HLA-A2 patients with
malignant gliomas, we have examined whether peptide-analogues can be
used for optimal expansion and activation of IL-13R2 specic CTL. We
synthesized three IL-13R2 (345-353)-analogue peptides by substitutions
of the carboxyl terminal isoleucine (I) for valine (V) and the amino terminal
tryptophan (W) for either alanine (A), glutamic acid (E) or non-substituted
(W) (designated as IL-13R2-1A9V, -1E9V, and -9V, respectively). Among
these, the modied peptides IL-13R2-9V and -1A9V displayed higher
binding afnity to HLA-A2 than the native IL-13R2 (345-353) epitope.
Stimulation of HLA-A2 glioma patient-derived CD8 T cells with autolo-
gous dendritic cells (DCs) loaded with IL-13R2-9V and -1A9V induced
higher levels of lytic activity against T2 cells loaded with low concentrations
(0.110 nM) of native IL-13R2 (345-353) peptide than other peptides
including the native IL-13R2 (345-353). These CTL lines induced with
IL-13R2-9V or -1A9V peptides lysed HLA-A2 glioma cells that express
IL-13R2 more efciently than CTL induced by the wild-type peptide.
Both IL-13R2-9V and -1A9V demonstrated superior immunogenicity to
the native peptide at equal levels in most cases. However, we have observed,
in multiple HLA-A2 glioma patients, that IL-13R2-9V is the most con-
sistent and strongest inducer of CTL responses against IL-13R2 glioma
cells. In order to examine whether immunization with IL-13R2 (345-353)

and/or its peptide analogues can elicit CTL responses in vivo, we employed
HLA-A2 transgenic mice (HHD mice). Immunization of HHD mice with
IL-13R2-9V or -1A9V peptides induced higher levels of CTL reactivity
in their splenocytes against the EL4-HHD cells pulsed with IL-13R2
(345-353) peptide as well as the EL4-HHD cells endogenously expressing
IL-13R2 (EL4-HHD-IL-13R2) in comparison to IL-13R2 (345-353).
These ndings indicate that highly antigenic IL-13R2 peptide-analogues
may be useful for the development of vaccines capable of effectively expand-
ing IL-13R2 specic CTL in glioma patients. We are currently evaluating
the anti-tumor effect of immunizations with IL-13R2-9V or -1A9V pep-
tides in HHD mice bearing EL4-HHD-IL-13R2 tumors.
357. ENHANCED ANTI-TUMOR IMMUNE RESPONSE
AFTER SELECTIVE INHIBITION OF INDUCIBLE NITRIC
OXIDE SYNTHASE IN RATS CARRYING INTRACEREBRAL
TUMORS AND IMMUNIZED WITH IFN- GAMMA
SECRETING GLIOMA CELLS
W. Badn, P. Hegardt, M. Fellert, A. Darabi, M. Esbjrnsson, E. Visse, S.
Janelidze, L. Salford, and P. Siesj; Clinical Neurosurgery, Neurosurgery,
Lund, Sweden
In this study we aimed to clarify whether IFNg could enhance anti-
tumor immune responses. We investigated both an iNOS selective, L-N
6
-
(1-iminoethyl)-l-lysine (L-NIL), and a non selective, N-nitro-l-arginine
methyl ester (L-NAME), inhibitor of NOS. Both L-NIL and L-NAME were
able to inhibit NO production and enhanced the proliferation and produc-
tion of IFN-g from polyclonally activated spleen cells in vivo. However,
L-NIL had a broader window of efcacy and a lower minimal effective
dose. In correlation to in vivo results, only spleen cells from rats treated
with L-NIL, and not L-NAME, had an enhanced proliferation as well as
IFN-g production in response to tumor cells. Furthermore, L-NIL was able
to prolong the survival of rats with intracerebral tumors after therapeutic
immunizations with tumor cells secreting IFN-g. These results imply that
selective inhibition of iNOS can enhance anti-tumor immune responses
evoked by IFN-g treatment.
358. ALTERATION IN THE TH1 AND TH2 CYTOKINE
PROFILE IN PATIENTS WITH BRAIN TUMOURS
D. Kamdar,
1
R. Kumar,
2
J. Greenman,
1
D. OBrien,
2
and L. Madden
1
;
1
Medical Research Division, University of Hull;
2
Department of
Neurosurgery, Hull Royal Inrmary, Kingston Upon Hull, UK
The objective of this study was to evaluate the TH1 and TH2 cytokine
prole in patients with brain tumors. IL-12 is a key cytokine mediating a
TH1-type response, and IL-10 is a TH2-type pleiotrophic cytokine. These
two cytokines act in an antagonistic fashion, with a balance point that var-
ies between the physiological and pathological state (OHara et al., Clin.
Cancer Res. 4, 1943, 1998). The study was designed as a prospective analy-
sis to compare changes in serum IL-10 and IL-12 levels in patients with
newly diagnosed brain tumors. An initial cohort of 65 patients aged 24 to
78 years were recruited between December 2002 and December 2003 fol-
lowing radiological diagnosis of a space-occupying lesion. Serum samples
were obtained prior to surgery. Age and sex matched control patients (n
36) were also recruited. Serum IL-10 and IL-12 were measured by quan-
titative ELISA (BioSource International.). There were 12 benign, 5 low-
grade, 30 high-grade, and 18 metastatic tumors. Serum IL-10 was detected
in only 47% of the controls (median [m] 0.0 pg/ml). IL-10 was detected in
92% of patients with benign tumors (m 2.4 pg/ml), 60% of low-grade
tumors (m 0.0 pg/ml), 67% of high-grade tumors (m 0.1 pg/ml), and
78% of metastatic tumors (m 0.3pg/ml). Conversely, serum IL-12 was
detected in all patients in the control group (m 52.2 pg/ml). However,
IL-12 was only detected in 67% of benign tumors (m 12.1 pg/ml), 80%
of low-grade tumors (m 41.1 pg/ml), 27% of high-grade tumors (m
0.0 pg/ml), and 33% of metastatic tumors at 0.0 pg/ml. The only factor
that showed statistical signicance was serum IL-12 levels where there was
reduction in the high grade and metastatic group compared with controls
(P 0.001). The TH1-like cytokine IL-12 decreased signicantly as tumor
stage progressed; however no increase in the antagonistic TH2-like cyto-
kine IL-10 was observed.
359. MODIFICATION OF TUMOR MICROENVIRONMENT
BY DELIVERY OF CYTOKINE- GENE TRANSFECTED
DENDRITIC CELLS ENHANCES THE EFFICACY OF TYPE-1
ANTIGEN-SPECIFIC T CELL ADOPTIVE TRANSFER
F.N. Nishimura,
1
J.E.D. Dusak,
1
M.H. Hatano,
1
J.E. Eguchi,
1
W.G.
Gao,
2
A.G. Gambotto,
2
W.J.S Storkus,
3
F.M. Monzon-Bordonaba,
4

J.L. Lyons-Weiler,
4
and H.O. Okada
1
;
1
2
Surgery,
3
Dermatology, and
4
Microarray and Analysis Shared Services and
Bioinformatics Shared Services, University of Pittsburgh Cancer
Institute, Pittsburgh, Pennsylvania, USA
We have previously demonstrated that injection of murine central
nervous system (CNS) tumors with dendritic cells (DC) secreting inter-
feron (IFN)-a (DC-IFNa) remarkably enhanced the therapeutic effect of
peripheral vaccinations with ovalbumin (OVA)-specic T cell epitopes. The
injected DC-IFNa migrated from the CNS tumor site to the draining cervi-
cal lymph nodes (CLNs), where they cross-primed tumor antigen-specic
CTLs (Okada et al., 2004). To further determine the effect of this prime
and boost vaccine-strategy on the effector cells, we employed adoptive
transfer of OVA-specic type I CD8 effector T (Tc1) cells derived from
OVA-specic T cell receptor transgenic OT-1 mice. In addition, to further
understand molecular events induced by the IFN-a delivery in the CNS
tumors, we analyzed the gene-expression proles within the tumor micro-
environment using Codelink Mouse 20K microarray system. Syngeneic
C57BL/6 mice bearing day 5 OVA-transfected B16 melanoma (M05) in
the CNS received intratumoral (i.t.) delivery of DCs ex vivo transfected
with adenoviral murine IFN-a or control vector y5. On day 6, these mice
also received intravenous injections with 5 10
6
Tc1 cells. DC-IFNa
induced expression of IFN-inducible protein (IP)-10, which is a potent
chemokine for type I immune effector cells, from M05 cells. Tc1 cells also
expressed CXCR3, which is a primary receptor for IP-10 and a high level
of CD44 activation marker. As expected from these observations, mice
treated with i.t. DC-IFN-a and i.v. Tc1 injections resulted in prolonged
survival of the host animals and accumulation of higher numbers of OVA-
tetramer/CD8 cells in the cervical lymph nodes in comparison to the
control treatment mice that received i.t. DC-y5 and i.v. Tc1. Microarray
analyses of the CNS tumors indicated that local IFN-a expression upregu-
lated a variety of immuno-regulatory molecules such as major histocompat-
ibility complex (MHC) class I chains, a DC maturation factor 2,5 oligoad-
enylate synthetase and T cell activation marker CD69, suggesting that local
IFN-a expression enhances antigen presentation processes and activation of
effector T cells within the tumor microenvironment. In addition, as inter-
leukin (IL)-23 has been recognized as a critical mediator of chronic inam-
matory responses in the CNS, we evaluated the effect DC-mediated delivery
of IL-23 to the CNS tumors. Although IL-23 did not induce IP-10 from the
tumor cells or IFN-g from the Tc1 cells, tumor inltrating lymphocytes
from IL-23 and Tc1 treated mice demonstrated a remarkably increased
number of OVA-tetramer/CD44 cells in comparison to the control and
IFN-a treated animals, suggesting that IL-23 has a unique ability to attract
antigen-specic effector cells to the tumor via distinct mechanisms than
IFN-a. These results warrant further evaluation of a combined approach
with IFN-a and IL-23 delivery to CNS tumors.
360. PILOMYXOID ASTROCYTOMA: A RARE BUT
CLINICALLY SIGNIFICANT SUBGROUP OF OPTIC
PATHWAY GLIOMAS
G. Kammler,
1
C. Hagel,
2
K. Bentele,
3
K. Ittner,
3
U. Grzyska,
4

K. Lamszus,
1
M. Westphal
1
; Departments of
1
Neurosurgery,
2
Neuropathology,
3
Pediatrics, and
4
Neuroradiology, University Hospital
Hamburg-Eppendorf, Hamburg, Germany
Optic pathway/hypothalamic gliomas occur mostly in children, belong
to the histological subgroup of pilocytic astrocytomas (PA), and take highly
variable clinical courses during adolescence and later in adulthood. It has
been stated that the pilomyxoid variant (pmPA) is more aggressive and
makes up a high percentage of PAs. We review a series of 59 cases of recent
pediatric and adult cases including three pmPAs and specically focus on
one particular case of pmPA and its remarkable clinical and neuroradiologi-
cal course. Between 2000 and 2004, 37 children and 22 adults were oper-
ated with intracranial PAs of which 12 were optic pathway PAs (20.1%).
Of the 8 pediatric cases, 3 were limited to the optic nerves, chiasm, and ret-
rochiasmatic extracerebral aspect of the optic tract. Two cases expanded
into the hypothalamus/third ventricle, and three were spread through the
hemisphere. In the adult population, only two cases were related to the
optic system, one being optochiasmatic with extension into the third ven-
tricle and one originating from the optic tract with mainly temporal exten-
sion. As for the neuroradiological growth pattern, three groups of tumors
can be distinguished in our group: (1) solid, non-cystic, mostly extrace-
rebral tumors, (2) cystic intraparenchymal/ intraventricular tumors, and
(3) solid, non-cystic inhomogenously enhancing tumors. A case of pmPA
was found among the latter group, diffusely spreading from throughout
the hemisphere and the brainstem. Within this tumor, strongly enhancing
nodules with rapid growth appeared at different times and places requiring
transylvian and transventricular decompression, respectively, for neuro-
logical symptoms. These nodules both turned out to be of the pilomyxoid
subtype and also during surgery were clearly distinct, being of a greenish
jelly-like texture. Complete removal of these nodules resulted in stable local
control while new nodules rapidly occurred. The pilomyxoid variant of
PA seems to harbor cell clones that rapidly expand despite absence of any
signs of anaplasia. They appear to have limited invasive capacity because
even within a very diffuse tumor, they can be locally controlled by surgery.
The highly diverse behavior of the different compartments of this tumor
allow recommendation of aggressive treatment either by microsurgery or
interstitial radiosurgery for these spherical nodules which determine the
poorer outcome of these cases.
361. DUMBBELL TRIGEMINAL SCHWANNOMA IN
A CHILD: COMPLETE REMOVAL BY A ONE-STAGE
PTERIONAL SURGICAL APPROACH
C.P. Verstappen, T. Beems, and E.J. van Lindert; Neurosurgery,
University Medical Centre Nijmegen, Nijmegen, The Netherlands
The objective of our study was to describe a rare case of a trigeminal
schwannoma in a child and the surgical procedure performed for therapy.
A six-year-old girl presented with tiredness, pain in the neck region, gait
disturbances, and dysarthric speech. She did not have any skin manifesta-
tions like caf au lait spots or subcutaneous tumors. Family history did
not suggest familial neurobromatosis. Imaging studies revealed a unilat-
eral dumbbell-shaped tumor, both extending in the middle and posterior
fossa, centered over Meckels cave. A one-stage surgery was performed by
pterional craniotomy. The tumor was rst debulked in the middle fossa,
then peeled from the wall of the cavernous sinus, followed by extirpation
of the tumor from the posterior fossa. The tumor extended to the caudal
cranial nerves, and was completely removed. Trigeminal fascicles were dis-
tributed throughout the tumor. Histopathological examination revealed
a schwannoma. Trigeminal schwannoma is a rarely occurring tumor in
childhood, only nine cases having been reported in the literature thus far.
Furthermore, although several often multi-staged surgical strategies for
dumbbell tumors are described, in this patient the tumor was eradicated by
a one-stage pterional approach.
362. ATYPICAL TERATOID/RHABDOID TUMORS: A
NEW MALIGNANT PEDIATRIC BRAIN TUMOR TO BE
DISTINGUISHED FROM MEDULLOBLASTOMA/PNET
H. Hidehiro and F. Kiyotaka; Neurosurgery, Kitasato University,
Sagamihara, Japan
The clinicopathological features of atypical teratoid/rhabdoid tumor
(AT/RT), a new entity among malignant pediatric brain tumors, and the
differential diagnosis from primitive neuroectodermal tumor (PNET)/
medulloblastoma are described. The clinicopathological features of 140
AT/RTs including 7 Japanese cases, 70 cases of Mayo Clinic and Ameri-
can Pediatric Oncology Group, and 63 published cases subject to analysis
included patient age and sex at presentation, symptoms, neurological signs,
tumor location, histopathological features and outcome. The patients with
AT/RT aged from 22 days to 14.9 years. There was a 1.5:1 male predomi-
nance. The clinical symptoms were related to tumor location and usually
nonspecic in nature. Of the 140 AT/RTs, 61% were located in the poste-
rior fossa, 20% in the cerebral hemispheres, 5% in the suprasellar and/or
third ventricular regions, 5% in the pineal region. Histologically, AT/RT
is dened as a polymorphous neoplasm often featuring rhabdoid, PNET,
epithelial, and mesenchymal components. AT/RTs usually include PNET
components and occur mainly in the posterior fossa, so mimic medullo-
blastoma. AT/RT is characterized by the cytogenetic nding of monosomy
22 rather than i(17q). The tumor is similarly mistaken for PNET at supra-
tentorial sites. Germ cell tumors also enter into the differential diagnosis
due to their histological immunophenotypic diversity, particularly features
indicative of epithelial and mesenchymal differentiation. Nonetheless, the
remarkable spectrum of tissues that typify teratoma is absent in AT/RT.
The prognosis of AT/RT is far less favorable than that of PNET/medul-
loblastoma of malignant or germ cell tumor. Meta-analysis of 140 cases
conrms that AT/RT is as a clinicopathological entity and emphasizes the
necessity for distinguishing this unique tumor from other pediatric central
nervous system neoplasms.
363. THERAPEUTIC EFFICACY AND PROGNOSTIC
FACTORS IN MEDULLOBLASTOMAS
T. Ota,
1
T. Watanabe,
1
J. Kurihara,
2
A. Yoshino,
1
Y. Katayama,
1

H. Nishimoto,
2
and H. Kishimoto
3
;
1
Neurological Surgery, Nihon
University School of Medicine, Tokyo;
2
Neurological Surgery and
3
Pathology, Saitama Childrens Medical Center, Saitama; Japan
Medulloblastomas are the most common central nervous system malig-
nancies in children. Combined modality treatment with radiation and che-
motherapy has substantially improved disease control and extended patient
survival. Nevertheless, medulloblastoma remains a treatment challenge, and
considerable controversy exists as to the best therapeutic management for
patients with such tumors. In the present study, we retrospectively analyzed
a series of 21 patients with newly diagnosed medulloblastomas treated by
surgery, radiation, and adjuvant chemotherapy. For the entire study popula-
tion, the median overall survival (OS) was 56 months, with 5-year OS rate
of 47%, and the median disease-free survival (DFS) was 41 months, with
5-year DFS rate of 44%. Radical surgery, low-stage according to the Chang
Staging System, and platinum chemotherapy were signicantly associated
with longer DFS and/or OS. Patients who were less than 3 years of age
exhibited a tendency toward a shorter survival. None of the immunohis-
tochemical markers including GFAP, S-100, NSE, synaptophysin, TrkA,
TrkC, and MIB-1 revealed a correlation with the survival. Our data indi-
cate that despite optimal treatment with surgery, radiation therapy, and
adjuvant chemotherapy, the prognosis for patients with medulloblastoma
remains dismal. Further clinical studies will need to address the exploration
of more aggressive therapeutic strategies in combination with conventional
craniospinal irradiation and intensive platinum-based chemotherapy.
364. PEDIATRIC OLIGODENDROGLIOMA IN THE MOTOR
STRIP
J.V. Anton,
1
M. Prelog,
2
H. Zunterer,
3
H. Maier,
4
E. Grasbon-Frodl,
5

W. Eisner,
1
T. Fiegele,
1
L.B. Zimmerhackl,
2
J. Burtscher,
1
and K.
Twerdy
1
;
1
Neurosurgery,
2
Pediatrics,
3
Radiology II, and
4
Pathology,
Medical University Innsbruck, Innsbruck, Austria;
5
Centre for
Neuropathology and Prion Disease, Ludwig-Maximilians-University
Munich, Klinikum Grohadern, Munich, Germany
We present the case of an eight-year-old boy who presented with focal
seizures. MRI showed a mass (diameter 4 cm) in the left motor cortical area
with slight contrast enhancement on the oor of left sulcus centralis and no
contrast enhancement in the motor strip. Subtotal surgical resection was
guided by intraoperative ultrasound and intraoperative CT. Direct bipolar
cortical electrophysiological mapping informed the neurosurgeon about the
motor functionality of the exposed structures, which were inltrated by
tumor tissue. Finally, the contrast medium enhanced tumor part could be
resected due to combined use of intraoperative imaging and intraoperative
electrophysiology. Tumor within the motor strip was spared to avoid func-
tional impairment. Although Ki-67 staining showed elevated proliferative
activity, a low-grade oligodendroglioma (WHO grade II) was diagnosed
after histomorphological examination. Loss of heterozygosity (LOH) anal-
ysis was carried out using microsatellite markers and PCR based assay. No
allelic losses on chromosome arms 1p and 19q were shown. Post-surgery
the boy presented with paresthesia and mild motoric function disability
of the right hand, which diminished after a few days. The focal seizures
of the right arm persisted after surgery. Postoperative MRI (after 3 and 11
months) demonstrated a stable, remaining T2 extension in the motor strip.
There were no signs of tumor progression. As a consequence, the patient
received no further adjuvant chemotherapy.
365. PEDIATRIC NEURO- ONCOLOGY FROM 2500 BC TO
NOW: A HISTORICAL PERSPECTIVE
D. Walker,
1
C. Bailey,
2
R. Taylor,
2
G. Perilongo,
3
and J. Punt
1
;
1
Childrens Brain Tumor Research Centre, University of Nottingham,
Nottingham, UK;
2
Department of Paediatric Oncology, St James
Hospital, Leeds, UK;
3
Department of Paediatric Oncology, University of
Padova, Padova, Italy
Pediatric neuro-oncology has evolved as an international subspecialty
since the establishment of the International Symposium of Paediatric
Neuro-Oncology in 1985. Historically, the scientic body knowledge which
forms the basis for modern practice can be traced to 2500 BC. Studying
the evolution of this specialty identies development of scientic discover-
ies, technical inventions, and combined modality working from the late
nineteenth century to the present day. Consultation of a wide variety of
texts, Web sites, experienced colleagues, and other historical sources helped
create a historical timeline, depicting events, people, institutions, journals,
scientic advances, awards and political events that have shaped the current
state of knowledge and practice. This information will be used to stimulate
further contributions of important historical events, nationally and inter-
nationally, by a request to delegates to identify additional events as part of
an ongoing chronology of events that will evolve. The rst draft has already
been incorporated in Brain & Spinal Tumors of Childhood, Editors, D.A.
Walker, J.A.G. Punt, R. Taylor, and G. Perilongo (Arnold). A summary of
the timeline will be presented of the evolution of neuroscience, neurosurgi-
cal practice, institutions, diagnostic imaging, radiotherapy, chemotherapy,
cancer science and epidemiology that have been identied so far as making
major contributions to the clinical and scientic practice of neuro-oncology.
Further collection and subsequent dissemination of information from this
timeline will stimulate international debate of the national and interna-
tional scientic and clinical developments critical to establishment of effec-
tive clinical services for children with brain and spinal tumors in our respec-
tive national health systems.
366. MALIGNANT CENTRAL NERVOUS SYSTEM TUMORS
IN CHILDREN UNDER 3 YEARS OF AGE: SINGLE
INSTITUTIONAL EXPERIENCE
C. Wynne, S. Mackie, and J. Sastry; Paediatric Oncology, Southampton
General Hospital, Southampton, UK
It is perceived that the prognosis is poor in central nervous system
(CNS) tumors diagnosed in children less than 3 years of age. Surgery and
chemotherapy are the mainstays of treatment in this group of patients.
Radiotherapy is rarely used as a modality of treatment, primarily due to
concerns about its effects on brain growth and neurodevelopmental out-
come. The purpose of this study was to review the types of tumors, mode of
presentation, and management strategies used, as well as to assess outcome.
Patients under the age of 3 years diagnosed with CNS tumors between 1
September 1994 and 31 August 2004 were identied retrospectively from
the database in a UKCCSG pediatric oncology centre. Analysis of man-
agement and outcome was performed using patient records. Thirty-four
patients were identied, of which 18 were male and 16 female. The median
age at diagnosis was 20.8 months (interquartile range, 12.428.5 months).
The pathological diagnosis was available in 32 patients: primitive neuro-
ectodermal tumor (PNET) (12 patients), astrocytoma (8 patients), optic
glioma (2 patients), tectal glioma (2 patients), brain stem glioma (1 patient),
oligodendroglioma (1 patient), choroid plexus papilloma (2 patients), epen-
dymoma (2 patients), gangliocytoma (1 patient), and pineoblastoma (1
patient). Two additional patients with CNS tumors had no pathological
diagnosis. Six patients were treated with surgery alone, 5 patients with che-
motherapy alone, and 7 patients with both modalities. Surgery, chemother-
apy, and radiotherapy were used to treat 1 patient with optic glioma and 1
patient with ependymoma. The patient with brain stem glioma was treated
with radiotherapy alone. Five patients, including 1 with PNET, received
no active treatment and remain alive. Six patients with PNET and the 2
patients without pathological diagnoses died, having not received active
treatment. At the end of the 10-year period, 19 patients overall were still
alive. Of the children who had not survived, 11 had PNET, 1 had astrocy-
toma, and 2 did not have a pathological diagnosis. There was no apparent
trend of improving survival in the last 5 years. In this local group of patients
diagnosed with a CNS tumor below the age of 3 years, 19 out of 34 patients
(55%) are alive at the time of the study. The prognosis remains poor, but
this appears to be largely attributable to the high mortality associated with
PNET, the most common CNS tumor in this series.
367. CHRONIC LOW-DOSE CHEMOTHERAPY FOR
REFRACTORY OR RECURRENT BRAIN TUMORS OF
CHILDHOOD
T. Yanagisawa,
1
M. Akiyama,
1
K. Yokoi,
1
Y. Yuza,
1
Y. Kato,
1

K. Fujisawa,
1
T. Joki,
2
S. Ohi,
2
and Y. Eto
1
;
1
Paediatric Oncology;
2
Neurosurgery, Jikei University School of Medicine, Tokyo, Japan
Prolonged low-dose chemotherapy, which exploits the schedule depen-
dency of the agents, has sometimes demonstrated increased activity in both
hematological and solid refractory malignancies of childhood compared
with bolus administration. This makes it a reasonable choice for palliative
treatment in children who are otherwise incurable. A prospective phase
2 study has been conducted in our institute to evaluate the efcacy and
toxicity of prolonged schedule oral etoposide in children with refractory
or relapsed malignancies in a palliative treatment setting. Patients were
treated with oral etoposide (50 mg/m
2
per day given daily for 21 consecu-
tive days every 4 to 5 weeks) after failing in medical, radiological, and/or
surgical treatments. End point for follow-up was disease progression. Four
patients with brain tumors were entered. Patient 1 was a 6-year-old boy
with suprasellar mature teratoma with malignant component. Two years
after the initial treatment with repeated surgeries, radiotherapy, and chemo-
therapy, he had local relapse. Failing in ICE and BEP regimen chemotherapy
and gamma-knife treatment, oral etoposide was initiated for progressive
tumor. Stable disease duration was 28 months with oral etoposide. Patient 2
was a 4-year-old girl with suprasellar immature teratoma. Six months after
high-dose chemotherapy with thiotepa and toposide regimen and PBSCT
for the rst local relapse, tumor progression was noticed. Oral etoposide
was initiated for the recurrent tumor after failing in ICE regimen chemo-
therapy. Progression-free survival was 11 months. Patient 3 was a 1-year-
old girl with pontine low-grade astrocytoma. Oral etoposide was begun
after failing in initial chemotherapy with vincristine and carboplatin. She
has been alive at home for 31 months without any signs of tumor progres-
sion. Patient 4 was a 13-year-old boy with brain stem tumor. Failing in
initial radiotherapy (54 Gy), oral etoposide was begun. Four months after
the treatment, MRI revealed tumor progression and weekly vinblastine (6
mg/m
2
) was initiated. He has been at home without tumor progression for 8
months. Chronic oral etoposide and weekly vinblastine appeared to be well
tolerated, had modest toxicity, and showed excellent palliative effect in the
children with refractory and progressive brain tumors. Further large-scale
investigation seems justied to determine the indication and the optimal
use of these treatments.
368. THERAPEUTIC STRATEGY FOR ATYPICAL TERATOID/
RHABDOID TUMOR: A REPORT OF TWO CASES AND
REVIEW OF THE LITERATURE
A. Gomi
1
, Y. Koizumi
1
, H. Suzuki
1
, T. Miyata
1
, E. Matsumoto
1
,
H. Fujii
1
, S. Sakurai
2
, K. Fuse
1
, and E. Watanabe
1
;
1
Surgical Neurology;
2
Pathology, Jichi Medical School, Tochigi, Japan
Atypical teratoid/rhabdoid tumor (AT/RT) of the central nervous sys-
tem is a highly malignant neoplasm that primarily affects children less than
5 years of age. Despite aggressive multimodality therapy including surgi-
cal resection, chemotherapy, and irradiation, prognosis is dismal. Here we
report two young AT/RT patients with extended survival. The rst patient
was a male, 11 months of age at presentation. The tumor was located in the
right cerebellar hemisphere without radiographic metastatic disease. Initial
therapy consisted of gross total surgical resection followed by 7 cycles of
mild chemotherapy (cisplatin and etoposide). No relapse occurred within
33 months of initial treatment. The second patient was a female, 24 months
of age at presentation. The tumor was located in the fourth ventricle with-
out radiographic metastatic disease. Initial therapy consisted of gross total
surgical resection followed by 3 cycles of mild chemotherapy (cisplatin and
etoposide). Relapse occurred within 3 months of initial treatment. Post-
recurrence treatment consisted of surgical resection and extended local
irradiation to a dose of 5600 cGy. After 17 months of continued remission,
cerebrospinal dissemination occurred. Following extended chemotherapy
(cisplatin/etoposide and ifosfamide), the size of the tumor was reduced.
After 4 months of continued remission, dissemination around the brainstem
recurred, and the patient died at 27 months of initial surgery. It is important
to consider the therapeutic strategy best suited to each individual AT/RT
patient. For children less than 3 years of age, we hesitate to use radiotherapy
because it impairs physical and mental development; therefore, extended
surgical resection and chemotherapy should be selected as initial therapies.
Some cases of AT/RT may not be sensitive to chemotherapy, and once we
have determined that chemotherapy is ineffective, we should not hesitate
to induce radiotherapy even if the child is less than 3 years old. Previous
reports have indicated that most AT/RT are radiosensitive. In the event of
recurrence, we recommend surgical resection and intensive chemotherapy.
369. RESIDUAL DISEASE IN INTRACRANIAL MALIGNANT
GERM CELL TUMORS (GCTS): FINDINGS GENERATED IN
SIOP CNS GCT 96 AND THEIR PROGNOSTIC IMPLICATION
G. Calaminus,
1
J. Nicholson,
2
D. Frappaz,
3
L. Garr,
4
C. Alapetite,
5
U.
Riccardi,
6
F. Saran,
7
T. Czech,
8
R. Kortmann,
9
and U. Gbel
1
;
1
Pediatric
Oncology, Hematology and Immunology, Heinrich-Heine-University,
Duesseldorf, Germany;
2
Pediatric Oncology, Addenbrookes Hospital,
Cambridge, UK;
3
Centre Leon Berard, Lyon, France;
4
Hematology/
Oncology, G. Gaslini Childrens Hospital, Genova, Italy;
5
Institut
Curie, Paris, France;
6
Radiation Oncology, Ospedale Infantile Regina
Margherita, Torino, Italy;
7
Radiotherapy, Royal Marsden Hospital NHS
Trust, Sutton, UK;
8
Neurochirurgie, Allgemeines Krankenhaus Wien,
Wien, Austria;
9
Radioonkologie, Universitt Leipzig, Leipzig, Germany
Residual disease is a predominant risk factor in most of the malignant
brain tumors. Therefore, the impact of residual tumor at the end of treat-
ment has been analyzed in patients with intracranial malignant GCTs who
are registered in the SIOP CNS GCT 96 trial. Residual disease is dened as
any abnormal contrast enhancement persisting in the former tumor region.
The patients under study are considered to be either germinoma patients
or non-germinoma patients. For the germinoma patients, after diagnosis,
treatment consists either of craniospinal irradiation 24 Gy/16 Gy tumor
boost (option A) or a chemotherapy with carboplatin/VP16/ifosfamide fol-
lowed by 40 Gy focal radiotherapy (RT) (option B). One hundred seven
patients are evaluated. MRI is assessed after operation, 3 months after RT,
and 1/2 year after diagnosis. Thirteen children have a complete resection
and are not analyzed for response. Fifty-four patients have a subtotal/par-
tial resection, of whom 39 receive a craniospinal RT (option A), and 15
children have a combined treatment (option B). A stereotactic biopsy has
been performed in 40 patients, of whom 31 receive option A and 9 children
option B. In summary, 30 patients have a residual lesion after RT between
0.2 and 2.0 cm in diameter. No patient undergoes a second operation or
other additional therapy. During follow-up, 13 tumors resolve, and only one
of 30 patients develops a progression. In comparison to the group without
residual lesion at the end of therapy (n 64, ve events) no difference
in event-free survival (EFS) can be detected (no residual 0.88 0.06 vs.
residual 0.97 0.03). For the non-germinoma patients, treatment after
diagnosis by markers and/or histology consists of either 4 courses PEI and
focal RT with 54 Gy or 30 Gy craniospinal RT/24 Gy tumor boost in case
of metastatic disease. One hundred fourteen children are evaluated. MRI
is assessed after operation, after chemo, and 3 months after RT. Thirty-six
patients show residual tumor after completion of therapy. In 26 patients,
no information about the status at the end of treatment is available. Of the
52 patients without residual tumor, 43 are in CR in contrast to 20 of the
36 children with residual disease. (EFS no residual 0.80 0.07 vs. residual
0.42 0.04). The impact of persisting tumor on prognosis in intracranial
malignant GCTs highlights the biological divergence of germinoma and
non-germinoma. Whereas in germinoma in the case of a residue an obser-
vant position is justied, in non-germinoma a surgical excision should be
performed whenever possible to decrease the high risk of recurrence. This
work was supported in part by Deutsche Krebshilfe.
370. MGMT AND p53 STATUS PREDICT TEMOZOLOMIDE
SENSITIVITY IN HUMAN MALIGNANT GLIOMA CELLS
M. Hermisson,
1
A. Klumpp,
1
W. Wick,
1
J. Wischhusen,
1
G. Nagel,
2

W. Roos,
2
B. Kaina,
2
and M. Weller
1
;
1
General Neurology, University
of Tuebingen, Tuebingen;
2
Toxicology, University of Mainz, Mainz;
Germany
Temozolomide (TMZ) is an alkylating agent which prolongs survival
when administered during and after radiotherapy in the rst-line treat-
ment of glioblastoma and which also has signicant activity in recurrent
disease. O
6
-methyl-guanine-DNA methyltransferase (MGMT) is a DNA
repair enzyme attributed a role in cancer cell resistance to O
6
-alkylating
agent-based chemotherapy. Using a panel of human glioma cell lines, we
demonstrate here that the levels of MGMT expression are a major predic-
tor of TMZ sensitivity in human glioma cells. The MGMT inhibitor, O
6
-
benzylguanine (O
6
-BG), sensitizes MGMT-positive glioma cells to TMZ,
whereas MGMT gene transfer into MGMT-negative cells confers protec-
tion. The anti-apoptotic BCL-X
L
protein attenuates TMZ cytotoxicity in
MGMT-negative LNT-229, but not in MGMT-positive LN-18 cells. Nei-
ther irradiation nor dexamethasone modulates MGMT activity or TMZ
sensitivity. Abrogation of p53 wild-type function by RNA interference or a
dominant-negative p53 mutant strongly attenuates TMZ cytotoxicity. Con-
versely, p53 mimetic agents designed to stabilize the wild-type conforma-
tion of p53 sensitize glioma cells for TMZ cytotoxicity. Collectively, these
results suggest that the determination of MGMT expression and p53 status
will help to identify glioma patients who will or will not respond to TMZ.
371. THE COMPARABLE EFFECT OF EXO- AND
ENDOGENOUS CANNABINOIDS ON APOPTOSIS IN
HUMAN MEDULLOBLASTOMA AND RAT GLIOMA CELLS
L. Storer,
1
A. Loku Kalutotage,
2
D. Walker,
1
and T. Parker
2
;
1
Department of Child Health;
2
School of Biomedical Sciences, University
of Nottingham, Nottingham, UK
Synthetic cannabinoids act by mimicking endogenous substances
(endocannabinoids) which activate specic cannabinoid receptors (Guz-
man, 2003). Cannabinoids have been shown to induce apoptosis, decrease
tumor growth, and inhibit tumor angiogenesis in a number of different
transformed cell lines and primary cells (Blazquez et al., FASEB J. 17, 529,
2003; Guzman, Nature Rev. 3, 745, 2003; Sanchez et al., FEBS Lett. 472,
39, 1998). In this study the effect of cannabinoids and endocannabinoids
was investigated on human medulloblastoma (Daoy) and rat glioma (C6)
cells. Cells were seeded in 6-well plates at a density of 50,000 cells/ml.
The apoptotic effect of cannabinoids and endocannabinoids was studied
by replacing the media after 24 h with either anandamide, 2-arachido-
nylglycerol (endogenous cannabinoid agonists), WIN-55,212-2 (CB
1
and
CB
2
agonist), Methandamide (CB
1
agonist) or AM281 (CB
1
antagonist).
Drug concentrations used were 10
-4
M, 5 10
-5
M, and 10
-5
M. Positive
controls were incubated with 10 M MG132, a known apoptotic inducer.
Cells incubated in the presence of DMSO or ethanol (carriers) provided
the baseline level of apoptosis. Cells were incubated for 3 and 7 days. Cells
were harvested and incubated with Annexin V and propidium iodide. The
number of live and the number of apoptotic cells were counted by using ow
cytometry. The results showed drug-dependent apoptosis after 3 days in 4
Daoy cells incubated with WIN-55,212-2 at 5 10
-5
M. However, there is
little drug-dependent apoptosis following 3 days incubation of Daoy and C6
cells with all other drugs tested. Trends show that after 7 days of incubation
the levels of apoptosis increased dramatically with all agonists. Treatment
with AM281 alone did not result in apoptosis at all concentrations. Control
experiments showed the carriers had no signicant effect on apoptosis at
concentrations lower than 5 10
-4
M on both cell lines. These results pro-
vide strong evidence to justify further investigation of exo- and endogenous
cannabinoids as therapeutic agents in the treatment of medulloblastoma
and glioma brain tumors.
372. LEVETIRACETAM (LEV) MONOTHERAPY IN PATIENTS
WITH PRIMARY BRAIN TUMORS (PBTS): EFFICACY, SIDE
EFFECTS, AND CSF AND PLASMA PHARMACOKINETICS
M. Previti,
1
M. Chamberlain,
3
S. Kesari,
4
P. Wen,
4
K. Edwards,
1

A. Van Horn,
1
and M. Glantz
2
;
1
Neuro-oncology, University of
Massachusetts, Worcester, Massachusetts;
2
Neurology, University of
Massachusetts Memorial Medical Center, Worcester, Massachusetts;
3
Neuro-oncology, University of Southern California, Los Angeles,
California,
4
Neuro-oncology, Massachusetts General Hospital, Boston,
Massachusetts; USA
LEV is a broad-spectrum antiepileptic drug (AED) with a novel mecha-
nism of action, excellent tolerability, no induction of the hepatic P-450 sys-
tem, and a half-life permitting twice daily dosing. These features suggest
that LEV would be an attractive AED for patients with PBTs, in whom
seizures are common and often poorly controlled, and in whom P-450 inter-
actions are a critical issue. Fourteen patients with PBTs and intraventricular
reservoirs for administration of intra-CSF chemotherapy received a single
oral loading dose of LEV at doses between 500 and 3000 mg. Plasma and
CSF LEV concentrations were measured serially between 0 and 48 h after
LEV administration. Data including epidemiologic features, seizure char-
acteristics, tumor type and therapy, concurrent medications, prior AEDs,
and dose, side-effects, and efcacy of LEV and any other AEDs was also
prospectively collected on 150 consecutive PBT patients receiving LEV
either as initial or replacement monotherapy for tumor-related seizures.
Loading doses of LEV of up to 3000 mg were well tolerated. The mean
T
max
of LEV in plasma was 5.2 h (range, 17), and in CSF 7.3 h (range,
315). The mean tof LEV in serum was 13.3 h (1420), and in CSF 24 h
(1340). No patients discontinued LEV secondary to adverse events. The
most common adverse events were mood disturbance (8%) and sedation/
fatigue (7%). Seventy-ve percent of patients had complete or good (1
seizure/month) seizure control with LEV, 83% of patients were receiving at
least one p-450 metabolized drug, and 76% of patients were treated with
at least one p-450 metabolized chemotherapeutic agent. Peak levels of LEV
are achieved rapidly in serum and CSF after a single oral loading dose. The
long t LEV in the CSF suggests a long duration of action and permits twice
daily dosing. Efcacy and tolerability are excellent, and P-450 interactions
are absent. LEV should be considered as rst- and second-line therapy in
patients with PBTs.
373. CHIMERIC PEPTIDES AS TUMOR-SELECTIVE
DELIVERY SYSTEMS
S. Jones and J. Howl; Molecular Pharmacology Group, School of Applied
Sciences, University of Wolverhampton, Wolverhampton, UK
The cell-type-specic targeting of cytotoxic agents and other functional
moieties can be achieved by using peptidyl address motifs that selectively
bind protein targets expressed at high density at the cell membrane. Indeed,
numerous studies have confirmed the utility of ligands for G protein
coupled receptors as components of heterofunctional peptide chimeras that
are selective biological probes. Our current efforts are directed toward the
further development of chimeric peptidyl constructs that employ sequences
derived from GPCR ligands or cell penetrant motifs to affect the selective
delivery of cytotoxins and signal transduction modulators to tumor cells.
We have designed and synthesized a range of hybrid constructs consisting
of cytotoxins (peptide and non-peptide) covalently linked to an address
peptide derived from the C-terminal of gastrin (G7; H-AYGWMDF-NH
2
).
The G7 homing motif targets a novel binding site expressed by U373MG
astrocytic tumor cells that is distinct from classical CCK
1
/CCK
2
receptors.
Moreover, biological responses following activation of this novel mem-
brane-bound protein may offer additional therapeutic advantages. For
example, G7 receptor activation is reported to inhibit the motility of malig-
nant astrocytoma in vivo while avoiding the growth-promoting effects of
gastrin (Pannequin et al., J. Pharmacol. Exp. Ther. 302, 274, 2002). We
evaluated the cytotoxicity of our chimeric peptides by comparing changes
in cellular viability using MTT conversion assays. Our data indicate that
chimeric peptides dose-dependently and rapidly (8 h) reduced the via-
bility of U373MG cells. Moreover, as a chimeric amino-terminal exten-
sion, the G7 address motif enhanced the cytotoxicity of both mastoparan
(H-INLKALAALAKKIL-NH
2
) and
D
(KLAKLAK)
2
peptides reported
to stimulate necrosis and/or apoptosis of eukarytoic cells. In conclusion,
hybrid G7 chimeras enhance the efcacy of cytotoxic agents and may be
valuable probes to investigate and manipulate additional aspects of astrocy-
toma cell biology. This work was supported by The Wellcome Trust.
374. INTRATUMORAL PHARMACOKINETICS
DETERMINED WITH MICRODIALYSIS IN A PATIENT WITH
GLIOBLASTOMA MULTIFORME FOLLOWING SYSTEMIC
ADMINISTRATION OF HIGH DOSE METHOTREXATE
J. Olson, J. Supko, S. Phuphanich, E. McKenzie, and S. Grossman;
NABTT CNS Consortium, Johns Hopkins Hospital, Baltimore,
Maryland, USA
Delivering potentially therapeutic concentrations of chemotherapeutic
agents to brain tumors remains a major concern in the design and inter-
pretation of clinical trials in neuro-oncology. This is the rst report of the
use of microdialysis in a human to monitor local concentrations of a sys-
temically administered anticancer drug within a brain tumor. This study
was conducted under a research protocol approved by the National Cancer
Institute and Institutional Review Board at each participating site. Patients
with recurrent and refractory glioblastoma multiforme (GBM) where a
surgical biopsy or partial resection followed by high-dose methotrexate
(MTX) was planned were eligible. A microdialysis catheter was placed
within residual contrast-enhancing tumor at the time of surgery. Modied
Ringers solution was delivered to the catheter at 1 ml/min. On the follow-
ing day, 12 g/m
2
of MTX (adjusted for renal function) were administered
as a 4-h intravenous infusion. Plasma and microdialysate were collected
at 30-min intervals from one hour before dosing to 24 h after completing
the infusion. MTX was assayed using high-performance liquid chromatog-
raphy with mass spectrometric detection, with a 0.5 ng/ml lower limit of
quantitation. The rst patient to be evaluated was a 51 year old who had
a biopsy to conrm tumor progression after prior surgery, radiation, Gli-
adel, and temozolomide. The plasma pharmacokinetics of MTX behaved as
expected. Renal function remained unchanged, and the leukovorin rescue
was administered as per protocol. MTX concentrations in extracellular
uid within the GBM rose rapidly to a maximum of 57 mM and subse-
quently decayed in a monoexponential manner with a half-life of 12.3 h. As
this was considerably slower than the loss of MTX from plasma, MTX con-
centrations in the dialysate actually exceeded the plasma levels beginning
at 16 h after the MTX infusion ended. The patient subsequently developed
urosepsis with neutropenia, but no mucositis or diarrhea, and died 3 weeks
later after care was withdrawn. Detailed review by NABTT, CTEP, and
the study site found no association with the microdialysis catheter and an
uncertain relationship to MTX. This initial experience has demonstrated
the ability to measure intratumoral concentrations of a chemotherapeutic
agent in a patient with a brain tumor using microdialysis techniques. This
approach could signicantly impact the selection of drugs, dosing sched-
ules, and routes of administration in clinical neuro-oncology trials.
375. A NOVEL MECHANISM OF PKA- AND PKC-
DEPENDENT DRUG RESISTANCE IN HUMAN GLIOMAS
INVOLVING PHOSPHORYLATION AND METABOLIC
ACTIVATION OF GLUTATHIONE S-TRANSFERASE P1
(GSTP1)
F. Ali-Osman,
1
H. Lo,
2
G. Antoun,
1
A. Friedman,
1
H. Friedman,
1

and D. Bigner
1
;
1
Surgery, Duke University Medical Center, Durham,
North Carolina;
2
Molecular Oncology, M.D. Anderson Cancer Center,
Houston, Texas; USA
The human GSTP1 protein, involved in phase II metabolism of many
anticancer agents used in brain tumor therapy and in the regulation of cell
signaling in response to stress, is highly expressed in a signicant propor-
tion of human malignant gliomas. The overexpression and nuclear localiza-
tion of GSTP1 has been associated with drug resistance and poor outcome
in gliomas and medulloblastomas. We report here a novel mechanism of
glioma resistance to chemotherapy that involves crosstalk between the
GSTP1 protein and two Ser/Thr protein kinases, PKA and PKC, frequently
activated in human gliomas, resulting in phosphorylation and signicant
enhancement of GSTP1 metabolic activity. Using cell-free systems and
MGR3 human glioblastoma cells, we showed both PKA and PKC to phos-
phorylate GSTP1 GSH-dependently, with a stoichiometry of 0.4 0.03 and
0.53 0.02 mole phosphate per mole GSTP1 protein. In the presence of
GSH, eight different PKC isoforms (a, bI, bII, d, e, g, h, and z), belonging to
the three major PKC subclasses phosphorylated the GSTP1 protein, albeit
with varying efciencies. Enzyme kinetic studies with GSTP1 proteins
mutated at candidate amino acid residues established Ser42 and Ser184 as
putative phospho-acceptor residues for both kinases in the GSTP1 protein.
The catalytic efciency, kcat/Km, of the phosphorylated GSTP1 was more
than double that of the unphosphorylated protein. In glioblastoma cells,
activation of PKA and PKC signaling resulted in almost a 3-fold increase
in specic GSTP1 activity and a signicant increase in the resistance of the
glioblastoma cells to BCNU, cisplatin, and 4-hydroxyifosphamide. These
ndings demonstrate PKA- and PKC-dependent phosphorylation of GSTP1
as a signicant post-translational mechanism of regulation of GSTP1 func-
tion. The GSH-dependence of the phosphorylation suggests that under
high intracellular GSH conditions, such as is present in most drug-resistant
tumors, the GSTP1 protein will exist in a hyperphosphorylated and meta-
bolically more active state. The increased resistance of glioblastoma cells
following activation of PKC and PKA and the associated increase in GSTP1
metabolic activity indicate the crosstalk between the Ser/Thr kinases and
GSTP1 as a novel mechanism of drug resistance in glioma cells. The nd-
ings also explain, in part, the high resistance of PKC-overexpressing glio-
mas to chemotherapy and suggest that PKC inhibitors have a potential to
overcome GSTP1-associated drug resistance in gliomas. This work was
supported by grants RO1 CA91438, RO1 CA79644, and P50-CA108786
from the NIH (USA).
376. ICOVIR-2, AN OLD FRIEND WITH A NEW FACE: E2F-1
MEDIATED GLIOMA SELECTIVITY FOR DELTA24-RGD
M. Alonso,
1
R. Alemany,
2
C. Gomez-Manzano,
1
W.K.A. Yung,
1
H.
Jiang,
1
and J. Fueyo
1
;
1
Neuro-Oncology, The University of Texas M.D.
2
Terapia Genica, Instituto
Catalan de Oncologia, Barcelona, Spain
Oncolytic adenoviruses are promising therapies for the treatment of
gliomas. However, the major hurdles that these treatments encounter are
selectivity and efcacy. Efcacy can be enhanced by broadening the tropism
of the adenovirus. In this regard, we increased the infectivity of the D24
adenovirus inserting and integrin-binding motif (RGD) into the adenovi-
ral ber. Specicity can be achieved by means of regulatory elements that
provide both cell-specic gene expression and viral replication in cancer
cells. In this study, we developed a system in which E2F-1 promoter drives
the expression of the mutant E1A, in the context of D24-RGD construct
(Icovir-2). We hypothesized that E2F1 promoter will increase the selectiv-
ity of Icovir-2 in gliomas due to the absence of a functional RB pathway
in these tumors and the low activity of E2F1 in normal brain. In order to
characterize the therapeutic potential of Icovir-2 we carried out in vitro
studies using two different gliomas cell lines, U-251MG and U-87MG,
expressing high and low CAR, respectively. Icovir-2 showed cytopathic
effect and replication capacity in both cell lines. Interestingly, restoration of
the Rb-pathway by means of transfer of pRB or p21 completely abrogated
the oncolotyc effect of Icovir-2. Importantly, restoration of the RB-pathway
in cells infected with D24-RGD showed partial rescue (50%) from the
cytotoxic effect. These data suggested an increased specicity of Icovir-2
for glioma cells in comparison to D24-RGD. Assessment of E1A levels by
Western blot revealed also reduced expression levels of the protein in cells
infected with Icovir-2 versus D24-RGD. Moreover, ber expression was
detected in D24-RGD but not in Icovir-2 infected cells. Importantly, Icovir-
2 was unable to replicate in arrested NHA, and the MTT analysis showed
a signicantly reduced cytotoxicity in comparison to D24-RGD. Expres-
sion levels of E1A in NHA were also substantially reduced in comparison
to NHA infected with D24-RGD. ChIP analysis showed that the cellular
E2F1 was able to bind and activate the Icovir-2 promoter and that transfer
of pRB resulted in transcriptional repression of E2F1 at the promoter loca-
tion by pRb. Notably, in vivo studies involving tail vein injection (5
10
vp)
followed by monitoring the levels of GPT (IU/L) showed that liver toxicity
induced by Icovir-2 was signicantly lower than toxicity induced by D24-
RGD. Our data showed that Icovir-2 presents improved therapeutic index
in glioma cell lines versus D24-RGD. We concluded that cancer-specic
promoters could complement tropism-enhancer strategies in the ght of
oncolytic adenovirus against cancer cells.
377. EFFECT OF DEXAMETHASONE ON THE CYTOTOXIC
EFFECT OF CLOMIPRAMINE IN HUMAN ASTROCYTIC
CELLS IN VITRO
S. Amar, K. Parker, R. Lisle, and G.J. Pilkington; Cellular & Molecular
Neuro-Oncology Group, School of Pharmacy & Biomedical Sciences,
University of Portsmouth, White Swan Road, Portsmouth, UK
Dexamethasone is a steroid frequently prescribed to patients with
primary high-grade brain tumors in order to control cerebral edema. Our
laboratories have already demonstrated that a tricyclic antidepressant, clo-
mipramine, selectively induces apoptosis in cultured brain tumor cells by
compromising the mitochondrial respiratory chain via complex III and con-
sequently activating a caspase pathway to cell death. It has previously been
also shown that dexamethasone inhibits and promotes apoptosis in vitro.
Clomipramine and dexamethasone are known to be interacting medica-
tions. Additive, synergistic, or inhibitory effects of dexamethasone on the
apoptotic effect of chlomipramine need therefore to be considered. In a series
of experiments IPSB-18 (passage 46), cultured anaplastic astrocytoma cells,
and UPESC (passage 11), a non-neoplastic astrocyte-rich population of cells
derived from the temporal lobe of an epileptic patient were exposed to clo-
mipramine (10 M) or dexamethasone (050 M) for 24 h. They were then
exposed to clomipramine (030 M) or dexamethasone (050 M) for
72 h (i.e., alternative clomipramine/dexamethasone and dexamethasone/
clomipramine treatment schedules). MTT assays were performed to assess
cell viability after drug exposure. We have also shown that dexamethasone
pretreatment of neoplastic astrocytes modulates oxygen utilization using a
Clarke Oxygen electrode assay and promotes apoptosis (Annexin V assay).
Pretreatment with clomipramine and subsequently with dexamethasone
showed no treatment-related effect in both cell lines. However, in tumor
cells, pretreatment with dexamethasone followed by clomipramine treat-
ment resulted in shift from cell death at 10 M (dexamethasone followed
by dexamethasone) to cell death at concentration 2 M. This effect was
not observed in non-neoplastic cells. Studies are currently in progress in
order to elucidate the molecular mechanisms underlying these effects. This
research is supported by the Samantha Dickson Research Trust.
378. CYTOKINES REGULATE INTERLEUKIN 13 RECEPTOR
ALPHA2 EXPRESSION IN GLIOMA CELLS
W. Debinski, N. Hu, and D.M. Gibo; Brain Tumor Center of Excellence,
Wake Forest University, Winston-Salem, North Carolina, USA
We documented that a vast majority of patients with high-grade gliomas
(HGG) overexpress IL13Ra2, a restricted receptor for interleukin13 (IL13)
and that this receptor is a very attractive target for anti-HGG molecularly
targeted therapies. We had suggested that epigenetic mechanisms involving
activating protein 1 (AP-1) activity are involved in the regulation of expres-
sion of IL13Ra2 in HGG, and recent studies revealed that the promoter
region of the receptors gene possesses AP-1 and also STAT-6 binding sites.
However, little is known how the expression of this receptor is biologically
regulated, and thus we conducted series of experiments in which HGG cells
were treated with either AP-1 or STAT-6 stimulatory cytokines, individu-
ally or in combination, and the levels of the receptor were monitored. The
IL13Ra2 protein levels were examined by Western blot and immunohisto-
chemistry (IH) in a variety of HGG cells and normal cells. Thus, the cells
were treated with epidermal growth factor (EGF) or IL4 or tumor necro-
sis factor alpha (TNFa), AP-1, and STAT-6 stimulants, respectively. We
have found that serum-starved HGG cells, such as A-172 MG, U-251 MG,
G48a, SNB-19, and U-87 MG, had the levels of immunoreactive IL13Ra2
decreased signicantly as detected by Western blotting and IH. This is sug-
gestive that IL13Ra2 is overexpressed in HGG in a nonconstitutive manner.
The addition of EGF or TNFa or IL4 to cells increased prominently the
levels of IL13Ra2 protein, usually by three- to tenfold, the extent of which
was cytokine-, cell line- and time-dependent. For example, EGF upregu-
lated the receptor most potently after 24-h treatment in the U-251 MG cells
and after 36-h treatment in G48a cells, while IL4 alone had little effect.
We next examined whether an already elevated IL13Ra2 can be further
upregulated by the studied cytokines in the presence of serum in HGG cells.
We found that supra-physiologic concentrations of EGF and TNFa, and less
that of IL4, could further increase the levels of the receptor in HGG cells.
The same experiments were performed on transformed normal glial cells
(SVGp12), human endothelial cells (HUVEC), and keratinocytes (HaCat).
In general, the background levels of immunoreactive IL13Ra2 were very
low in those cells when compared with HGG cells. EGF, TNFa, or IL4
produced an increase in the receptor levels, but the levels of IL13Ra2 were
still much lower than in HGG cells. IL13Ra2 is currently utilized preclini-
cally and clinically as a target for a variety of therapeutic approaches, such
as targeted recombinant cytotoxins, viruses, cytolytic T cells, and vaccines.
In this work, we demonstrate that a short-term pretreatment of HGG cells
with AP-1 and/or STAT-6 stimulatory cytokines should provide further
therapeutic advantage by upregulating the levels of IL13Ra2.
379. NATURAL SESQUITERPEN ALCOHOL ALPHA-
BISABOLOL, A NONTOXIC COMPOUND, STRONGLY
INDUCES APOPTOSIS IN GLIOMA CELL LINES
E. Cavalieri,
1
S. Mariotto,
1
L. Berra,
2
A. Carcereri de Prati,
1
A. Ciampa,
1

C. Fabrizi,
3
G. Lauro,
3
S. Leone,
3
R. Gottardo,
4
and H. Suzuki
1
;
Departments of
1
Neuroscience and Vision, Section of Biochemistry,
2
Neuroscience and Vision, Section of Neurosurgery, and
4
Medicine and
Public Health, Unit of Forensic Medicine, University of Verona, Verona;
3
Department of Biology, University of Roma Tre, Rome; Italy
Among human tumors, glioblastoma is one of the most malignant ones,
and despite aggressive surgical resection and radiotherapy, the median sur-
vival in these patients does not normally exceed 1 year (De Angelis, N. Engl.
J. Med. 344, 114, 2001; Surawicz et al., J. Neurooncol. 40, 151, 1998). The
use of systemic chemotherapy may improve the efcacy of treatment, but
its use is associated with signicant toxicity, and the long-term prognosis
remains poor (Parker et al., CA Cancer J. Clin. 46, 5, 1996). Carmustine
(BCNU) is, at concentration corresponding to LD10 (13 mg/kg), not able
to completely kill glioma cells in vivo (Rosenblum et al., J. Neurosurg. 58,
177, 1983). Numerous natural compounds have been reported to be poten-
tial anti-glioma agents, although major parts of these sank into oblivion.
a-bisabolol is a small, oily sesquiterpene alcohol with molecular mass of
222.37 Da, isolated from the essential oil of a variety of plants, shrubs, and
trees. Its toxicity in animals is very low (LD50 1314 g/kg, Merck Index).
In the present study, we report the cytotoxic effect and the type of death
induced by a-bisabolol in glioma cells. We examined, as a human glioma
cell model, T67 and U87 cell lines and, as an animal model, the rat glioma
cell line C6. At 2.53.5 mM, the viability of these cells was reduced to 50%
with respect to untreated cells in 24 h. Furthermore, the same concentra-
tions failed to affect the viability of normal rat astroglial cells, in line with
its reported non-toxicity in rats (Hernandez-Ceruelos et al., Toxicol Lett.
135, 103, 2002; Villegas et al., J. Nat. Prod. 64, 1357, 2001). At higher con-
centrations (10 mM) a-bisabolol killed completely the cells. Judging from
caspase 3 activation, poly(ADP-ribose) polymerase cleavage, DNA ladder
formation, and hypo-G1 accumulation, the cytotoxicity triggered by a-bis-
abolol results from the induction of apoptosis. Two major routes, extrinsic
and intrinsic, have been identied through which cytotoxic drugs induce
apoptosis. The rst one is mediated by death receptors. In the second path-
way, mitochondria play essential roles. The dissipation of mitochondrial-
inner transmembrane potential and the release of cytochrome c from mito-
chondria indicate that apoptosis occurs, in our experiments, through the
intrinsic pathway. Taken together, these results point out that a-bisabolol
may be considered a novel compound able to inhibit glioma cell growth
and survival.
380. ENHANCED EFFICACY AND MR IMAGING OF
CONVECTION-ENHANCED DRUG DELIVERY
Y. Mardor,
1
O. Rahav,
2
Z. Lidar,
2
A. Ocherashvilli,
1
D. Daniels,
1

Y. Roth,
1
S. Maier,
3
J. Zauberman,
4
and Z. Ram
2
;
1
The Advanced
Technology Center, Sheba Medical Center, Ramat-Gan, Israel;
2
Neurosurgery Department, Tel-Aviv Medical Center, Tel-Aviv, Israel;
3
Radiology, Brigham and Womens Hospital, Boston, Massachusetts,
USA;
4
Neurosurgery Department, Sheba Medical Center, Ramat-Gan,
Israel
Convection-enhanced delivery (CED) is a promising technique for dis-
tribution of drugs into brain and tumor tissue, currently being tested in
several phase 2/3 clinical trials. We have previously presented the clini-
cal application of diffusion-weighted MRI (DWMRI) for monitoring CED
of Taxol in recurrent GBM patients. CED is known to depend on several
physical and physiological parameters. After accounting for these variables,
initial clinical experience shows signicant variability in the extent of con-
vection among patients and among drugs. Therefore, increasing tumor
response to CED is essential, as well as real-time monitoring of the extent
of convection and its early effects on the tissue. Solutions containing com-
binations of Cremaphore, Taxol, carboplatin, ethanol, sucrose, and human
serum albumin in different concentrations were mixed with Gd-DTPA and
infused into normal rat striatum or intratumorally in rats bearing large 9L
tumors. T1 MRIs were acquired immediately post-treatment to assess the
extent of convection, and DWMRIs were acquired 24 h later to assess tis-
sue response. Some rats were monitored by MRI for an additional 4 days
to demonstrate subsequent formation of necrosis. The extent of convection
was reected by the T1 MRIs. Limited convection was characterized by sig-
nicant backow along the catheter and into the ventricles, while efcient
convection presented signicant spread into the striatum. CED with cyto-
toxic infusates was followed by signicant changes in subsequent DWM-
RIs. The extent of these changes correlated signicantly (Taxol, r
2
0.75, P
0.004) with the extent of convection as depicted by T1 MRIs. When con-
vection was not achieved, including with toxic infusates, no changes were
detected on DWMRIs. The efcacy and extent of convection were found to
correlate signicantly with infusate viscosity (r
2
0.73, P 0.003). While
low-viscosity infusates tend to backow, high-viscosity infusates tend to
form efcient convection. Increasing the viscosity of a carboplatin solution
with sucrose led to a signicant increase of infusate distribution within the
striatum, depicted by the immediate T1 MRIs (P 0.008). This effect was
accompanied by a corresponding increase in toxic tissue changes, depicted
by the later DWMRIs (P 0.007). In all 9L tumorbearing rats, DWMRI
revealed no tumor response, consistent with the immediate T1 MRIs, which
showed that the Taxol leaked out of the tumor and accumulated in normal
surrounding brain tissue. Our data suggest that Gd MRI and DWMRI can
be used for real-time assessment of CED efciency and early assessment of
tissue response. In addition, increasing the viscosity of solvents may be a
simple way to signicantly enhance the efcacy of CED treatments, thus
increasing their antitumor effect.
381. TARGETING MALIGNANT GLIOMAS WITH A LOW-
MOLECULAR-WEIGHT RAF/VASCULAR ENDOTHELIAL
GROWTH FACTOR RECEPTOR INHIBITOR
S. Sathornsumetee,
1
D. Batt,
3
M. Kieran,
2
S. Kier,
1
T. Ramsey,
3

N. Yusuff,
3
D. Reardon,
1
D. Bigner,
4
H. Friedman,
1
and J. Rich
1
;
Departments of
1
Surgery,
4
Pathology, and
5
Medicine, Duke University
2
Pediatrics, Dana-Farber
Cancer Institute, Boston, Massachusetts;
3
Oncology, Novartis Institutes
for Biomedical Research, Cambridge, Massachusetts; USA
Low-molecular-weight tyrosine kinase inhibitors against several growth
factor receptors have shown promise in preclinical malignant glioma stud-
ies, but more modest activity in clinical trials. We are currently investigating
novel intracellular kinases that may be frequently active in gliomas. Raf is
a critical intracellular mitogenic signaling mediator downstream from the
G-protein Ras family members that is occasionally mutated in gliomas.
Additionally, RAS and Raf activities are increased in most malignant glio-
mas through activation of growth factor receptor pathways. As multiple
growth factor receptors converge onto Raf, disrupting Raf function may
offer broad activity against tumors dependent on this pathway. AAL881 is a
novel, orally administered, small-molecule inhibitor of kinase activity asso-
ciated with Raf and vascular endothelial growth factor (VEGF) receptors.
We have now shown that AAL881 treatment of a highly resistant human
glioma cell line, D54MG, inhibits phosphorylation of downstream signal-
ing effectors, colony formation, VEGF secretion, and invasion through an
articial matrix. In addition, AAL881 inhibits cellular proliferation by
producing cell-cycle G
1
arrest without inducing signicant apoptosis. In in
vivo studies of athymic mice, AAL881 treatment was well tolerated without
signicant weight loss. A short-term (two-week) course of AAL881 treat-
ment in athymic nude mice with established subcutaneous D54MG xeno-
grafts not only signicantly delayed tumor growth in 5/10 mice, but also
cured 5/10 mice in repeated trials. Tumors less responsive to AAL881 treat-
ment displayed decreased proliferation relative to control tumors. AAL881
therapy in athymic mice with intracranial D54MG xenografts more than
doubled median survival compared to the control group. Of note, D54MG
xenografts display minimal sensitivity to VEGFR tyrosine kinase inhibi-
tors suggesting that either Raf plays an important role in tumor growth or
targeting combined Raf/VEGFR activity is highly active against this tumor.
Based on these results, Raf may represent a useful therapeutic target in glio-
mas. AAL881, a novel Raf/VEGFR kinase inhibitor, may offer a promising
therapy against malignant gliomas. This work was supported by grants
from Accelerate Brain Cancer Cure and the Pediatric Brain Tumor Founda-
tion of the U.S. J.N.R. is a Damon Runyon-Lilly Clinical Investigator and
a Sidney Kimmel Cancer Foundation Scholar.
382. COMBINATION THERAPY WITH PERIFOSINE
AND TEMOZOLOMIDE FOR GLIOMA TREATMENT:
PRECLINICAL TRIAL USING A MOUSE GLIOMA MODEL
H. Momota,
1
E. Nerio,
1
J. Finney,
1
Y. Lyustikman,
1
and E. Holland
1,2,3
;
1
Cancer Biology and Genetics,
2
Surgery (Neurosurgery), and
3
Neurology,
Memorial Sloan-Kettering Cancer Center, New York, New York, USA
Glioblastoma multiforme (GBM) is an incurable glial tumor even with
intensive therapy consisting of surgery followed by radio-chemotherapy.
Many newer forms of treatment have been tried for GBM, but these have
not been efcacious. Presenting, the best chemotherapeutic drugs for GBM
are alkylating agents such as carmustine (BCNU) and temozolomide. Alkyl-
phospholipid is in a novel class of antitumor agents, and perifosine is a
rst oral alkylphospholipid with a marked cytotoxic effect and fewer side
effects. Unlike most chemotherapeutic drugs that target the nuclear DNA,
perifosine interacts with the cell membrane and blocks signal transduc-
tion pathways. Recent studies have suggested the molecular mechanism of
perifosine action has the capacity to enhance radiation or other anticancer
drugs. However, the exact mechanisms of perifosines effect on gliomas are
still unclear. To develop a new paradigm for glioma therapy, we used mouse
glioma cell lines to investigate the mechanism of action of perifosine alone
and in combination with temozolomide. We also addressed this in vivo by
using a mouse glioma model. Mouse glial cells transformed with PDGF-B,
Kras, Akt, KrasAkt, and LacZ were used as an in vivo model. These cells
were treated with 300 M temozolomide and 45 M perifosine and ana-
lyzed with cell proliferation assay, Western blot, and cell cycle analysis. Gli-
omas ware induced in mice by PDGF gene transfer to the nestin-expressing
neural progenitor cells. Tumor-bearing mice were detected by biolumines-
cence imaging, and image-positive mice were treated with daily intraperi-
toneal administration of 100 mg/kg temozolomide and oral administration
of 30 mg/kg perifosine. Mice were then sacriced and tumor histology was
examined. Perifosine and temozolomide inhibited mouse glioma cell growth
in a dose-dependent manner, and these drugs had a synergistic effect in cell
culture. Cell cycle analysis showed a stronger G1 arrest with this combina-
tion rather than with each drug alone. In vivo, the mouse glioma model also
demonstrated reduction in tumor size by imaging and minimal staining for
proliferation markers by immunohistochemistry. Combination therapy of
perifosine and temozolomide is effective in a mouse glioma model and could
be a new candidate in treatment of human gliomas.
383. THE LUMINESCENT ATP ASSAY OR THE
COLORIMETRIC MTS ASSAYWHICH IS BETTER FOR
CHEMOSENSITIVITY TESTING IN MALIGNANT GLIOMAS?
R.V. Iyer,
1
T. Dawson,
1
K. Ashton,
1
C.H.G. Davis,
1
A. Golash,
1
P.
Roberts,
2
and R.W. Lea
2
;
1
Neurosurgery, Lancashire Teaching Hospitals
NHS Trust, Preston;
2
Biomedical Research unit, University of Central
Lancashire, Preston; UK
Chemosensitivity testing has been used clinically most successfully in
leukemia. Non-neurological malignancies provide large amounts of tumor
tissue for drug testing, while very little tissue is available in malignant astro-
cytomas due to limited surgery. There is a need for an assay methodology
sensitive enough to deal with small numbers of glial cells. Toward this end
we have developed a cell death assay by measuring ATP levels following
exposure of primary glial cells to different chemotherapeutic agents which
were compared with a tetrazolium dye reduction assay (MTS). Also, we
have investigated the role of apoptotic cell death by determining the level of
caspase enzyme activation within the cells. Astrocytic tumor tissue obtained
at surgery was disaggregated and plated in culture asks. At conuence the
cells were trypsinized and transferred to 96-well microtiter plates. At 70%
conuence, ve concentrations of each drug (cisplatin, BCNU, paclitaxel,
and etoposide) were added to the wells. After a period of 72 h, cell prolifera-
tion was measured by using the one-step MTS and ATP assays (Promega). A
separate luminescent microtiter plate was used to measure apoptotic activ-
ity by measuring caspase levels by using the one-step caspase assay (Pro-
mega). All tumors were either WHO grade III or IV astrocytomas. Assays
were performed at the rst passage, although the rate of cell growth was low
with some cultures limiting the plating density on the microtiter plates. A
high correlation (R
2
0.9) was observed between the ATP and MTS assays
when plating density per well was high (1000). However, the correlation
was poor (R
2
0.5) with low plating densities. There was variability in
the sensitivity of different primary cultures to the drugs used, with only
cisplatin showing a consistently toxic effect in the assays used. High levels
of caspase activation were only observed with paclitaxel and cisplatin at
the highest concentrations used, despite cell death being seen at lower con-
centrations. The luminescent ATP assay has a greater sensitivity to predict
drug response in glial cells, with lower cell numbers than the colorimetric
MTS assay, making it a potential tool for testing small biopsies. Estimation
of caspase levels show that at lower concentrations cisplatin and paclitaxel
may cause cell death through non-apoptotic mechanisms.
384. TISSUE INHIBITOR OF METALLOPROTEINASES-3
EXPRESSION IN THE CONTEXT OF AN ONCOLYTIC
ADENOVIRUS INHIBITS MATRIX METALLOPROTEINASE
ACTIVITY IN VIVO BUT DOES NOT ENHANCE ANTI-
TUMOR EFFICACY IN MALIGNANT GLIOMA
M. Lamfers,
1
D. Gianni,
2
C.H. Tung,
3
S. Idema,
1
P.H. Quax,
4
V.W.
van Beusechem,
5
W.P. Vandertop,
1
C.M.F. Dirven,
1
E.A. Chiocca,
2
and
W.R. Gerritsen
5
;
1
Department of Neurosurgery, VU University Medical
Center, Amsterdam, The Netherlands;
2
Molecular Neuro-Oncology
Laboratories, Neurosurgery Service and
3
Center for Molecular Imaging
Research, Massachusetts General Hospital, Harvard Medical School,
Charlestown, Massachusetts, USA;
4
Gaubius Laboratory TNO-PG,
Leiden, The Netherlands;
5
Department of Medical Oncology, Division
of Gene Therapy, VU University medical center, Amsterdam, The
Netherlands
A promising new approach to treatment of solid tumors is the use of
oncolytic viruses designed to replicate specically in malignant cells. We
hypothesized that insertion of a transgene encoding a secreted protein
which exerts effects on angiogenesis, apoptosis, and tumor cell inltration
would improve the anti-tumor activity of these agents. Tissue inhibitor of
metalloproteinases 3 (TIMP-3) constitutes an interesting transgene can-
didate for such an approach considering its reported inhibitory effects on
these processes. To assess the effects of TIMP-3 gene transfer to glioma
cells, we rst employed a replication-defective adenovirus encoding TIMP-
3 (Ad.TIMP-3). Infection of U-87MG, U-87dEGFR, and U-251MG glioma
cells with Ad.TIMP-3 inhibited in vivo invasion up to 86%. In addition,
Ad.TIMP-3 infection of a panel of primary glioma cell cultures obtained
from patients material decreased the viability of these cells and induced
morphological changes characteristic for apoptosis. On the basis of these
ndings, we proceeded to construct a conditionally replicating adenovirus
encoding TIMP-3. The TIMP-3 expression cassette was inserted into the
E3 region of the adenoviral backbone containing a 24 bp deletion in E1A.
This novel oncolytic adenovirus, AdD24TIMP-3, demonstrated enhanced
oncolytic activity on a panel of glioma cell lines and primary cell cultures
compared to the control oncolytic virus AdD24Luc. To conrm inhibition
of in vivo MMP activity by AdD24TIMP-3, nude mice bearing subcutane-
ous glioma xenografts received intratumoral injections of AdD24TIMP-3
or AdD24Luc. The functional activity of TIMP-3 was imaged noninvasively
by using a near-infrared uorescent MMP-2-activated probe. Tumoral
MMP-2 activity was signicantly reduced by 58.3% in the AdD24TIMP-3
treated tumors 24 h after infection. A study into the therapeutic effects of
combined oncolytic and antiproteolytic therapy was performed in both a
subcutaneous and an intracranial model for malignant glioma comparing
AdD24TIMP-3 to AdD24Luc. Treatment of subcutaneous (U-87MG) or
intracranial (U-87dEGFR) tumors with AdD24TIMP-3 and AdD24Luc
both signicantly inhibited tumor growth and survival compared to PBS-
treated controls. However, no signicant difference in anti-tumor activ-
ity between these oncolytic viruses was found. TIMP-3 was produced by
glioma cells infected with AdD24TIMP-3 in vivo and in vivo. The biologi-
cal effect resulting from an oncolytic adenovirus expressing TIMP-3 can be
imaged in vivo non-invasively demonstrating the functional expression of
the gene of interest. The anti-tumor effects of AdD24 could not be improved
by insertion of the TIMP-3 gene when injected during tumor development
in two murine models for malignant glioma.
385. OPTIMAL BIOLOGICAL DOSE AND SCHEDULE OF
INTERFERON ALPHA TO REDUCE THE GROWTH OF
HUMAN GLIOBLASTOMA IMPLANTED ORTHOTOPICALLY
IN NUDE MICE
D. Nam, M. Kim, and K. Park; Department of Neurosurgery, Samsung
Medical Center, Sungkyunkwan University School of Medicine, Seoul,
Korea
The purpose of this study was to optimize the antitumor activities of
pegylated IFN-alpha (PEG-IFN-alpha) against U87MG human glioblas-
toma cells growing orthotopically in nude mice. Twenty days after the intra-
cranial inoculation of tumor cells, groups of mice (n 5) were injected
with different doses of PEG-IFN-alpha (1,000, 5,000, 10,000, 25,000, and
125,000 units) twice per week subcutaneously. PEG-IFN-alpha at 10,000
units decreased the expression of basic broblast growth factor and matrix
metalloproteinase-2 most effectively. More than 25,000 unit injection did
not show the reduced expression of proangiogenic molecules by interferon.
Administration at the optimal biological dose (10,000 units, twice a week)
decreased tumor uptake (control: 6/6; PEG-IFN-alpha: 2/6) and progres-
sive growth of human glioblastoma cells. Mice that received chronic treat-
ment (4 weeks) reduced in tumor uptake and size compared with the short-
term treatment (2 weeks). With the immunohistochemical study, there was
signicant inhibition in the expression of proangiogenic molecules with
decreased microvessel density by PEG-IFN-alpha. The data suggest that
determination of optimal biological dose for PEG-IFN-alpha is important
in the clinical trial for glioblastoma patients.
386. PRECLINICAL FEASIBILITY AND SAFETY STUDY
OF A NOVEL ULTRASONIC DISPERSION SYSTEM FOR
INTRAPARENCHYMAL DRUG DELIVERY IN BRAIN
TUMORS
A.A. Kanner,
1
Y. Mardor,
2
H. Ezer,
1
L. Trejo,
1
Y. Wollman,
1
D.
Massad,
3
M. Goldman,
3
and Z. Ram
1
;
1
Neurosurgery, Tel Aviv Sourasky
Medical Center, Tel Aviv, Israel
We have developed a novel ultrasonic dispersion device (UDD) to treat
inltrative primary brain tumors by intra-tumoral delivery of therapeutic
molecules. The UDD consists of a dispersion wire within a guide tube. The
transmitter tip can be coated with a variety of particles of different size and
character. Ultrasonic pulses of different proles induce a high frequency
vibration (sonication) of the wire tip, which disperse particles from the
coated tip into the adjacent tissue. We conducted initial studies in a rat brain
model to investigate the local and remote effects of UDD on normal brain
tissue, and subsequently the feasibility and characteristics of in situ com-
pound dispersion of various compounds. The tested microparticles were
purple DS02/5290 (30 nm, Bangs Lab.), blue Fe
3
SO
4
(100 nm), and blue
Polystyrene (180 nm). The UDD probe was introduced stereotactically into
the right frontal lobe of male Fischer rats. The sonication was performed
with two different power proles as a single treatment cycle. Prole I lasted
for 60 s, source power 1 Watt with pulses of 1 s. Prole II lasted for 120 s,
source power 2 Watt with pulses of 2 s. After the procedure the probe was
removed and the brains were harvested after 30 min, 24 h, 96 h, and 10
days. Macroscopic and microscopic evaluation was performed on H&E
stained histological brain sections by a neuropathologist. Summary of the
research results: A total of 24 rats received a single cycle of treatment. The
stratication of the study groups was performed according to the two differ-
ent sonication treatment proles with 3 rats each per prole (2) and time
point (4). A signicant dispersion of the coating material (both solid and
suspension) within brain tissue was observed in all rats. Longer sonication
periods resulted in more extended distribution. During the post-treatment
observation period, no abnormal behavior of the study animals was evident.
Macroscopic and microscopic examinations of the brain tissue specimens
were negative for any evidence of tissue damage, cyst formation, or necrosis.
We have demonstrated the feasibility and safety of the UDD in a rat brain
model. Further efcacy studies in tumor-bearing rats are ongoing.
387. INCREASED ONCOLYTIC POTENCY OF THE
CONDITIONALLY REPLICATIVE ADENOVIRUS ADD24-P53
WHEN COMBINED WITH RADIOTHERAPY IN VIVO
S. Idema
1
, M. Lamfers,
2
S. Heukelom,
3
V. van Beusechem,
2

W. Gerritsen,
2
W. Vandertop,
1
and C. Dirven
1
; Departments of
1
Neurosurgery,
2
Medical Oncology, and
3
Radiotherapy, VU University
Medical Center, Amsterdam, The Netherlands
Conditionally replicative adenoviruses (CRAds) represent a promising
class of agents used against therapy-resistant brain tumors such as glioblas-
toma multiforme (GBM). The adenovirus Ad24-p53 has a 24 bp deletion
in the E1a region limiting replication to Rb mutant cells and encodes the
p53 tumor suppressor protein. It has shown superior oncolytic activity in
glioma compared to the parental control Ad24. As both replication com-
petent adenoviruses and exogenous p53 expression have been shown to
enhance radiosensitivity, the current experiments were performed to assess
the effect of Ad24-p53 and Ad24 in combination with radiotherapy in
GBM monolayer cultures, multicellular spheroids, and mouse xenografts.
U-87 and U-251 glioma cells growing in monolayers were irradiated with
3, 6, and 9 Gy. After 24 h, cells were infected at a multiplicity of infection
(MOI) of 0.1 or 0.01 with Ad24-p53 or the control Ad24. Eleven days
after treatment, viability was assessed by using a quantitative crystal vio-
let assay. U-87 spheroids were irradiated at 4 and 9 Gy and infected with
5 10
4
plaque-forming units (PFU) of Ad24-p53 or Ad24. Viability was
measured 12 days after infection using the tetrazolium saltbased WST-1
assay. U-87 subcutaneous mouse xenografts of approximately 170 mm
3
in
size were locally irradiated with 2 10 Gy on days 0 and 4 and received
4 10
8
PFU of Ad24-p53 on days 0, 2, and 4. Tumor size was measured
3 times weekly. The combination of Ad24-p53 and irradiation in U-87 or
U-251 cells synergistically increased cell kill with maximally 72% com-
pared to single treatments (ad24: 24-p53 and 9 Gy signicantly reduced
viability from 76% (infection alone) to 50% (ad24: 80% vs. 75%, respec-
tively). Combination treatment in U-87 mouse xenografts increased tumor
growth delay from 2 days (Ad24-p53) and 23 days (irradiation) to 32 days,
with 80% complete regression and 5/10 long-term survivors compared to
0/10 (irradiation) and 2/9 (Ad24-p53). Combining the radiosensitizing
effects of a conditionally replicative adenovirus and p53 expression has
the potential to greatly enhance the effect of radiotherapy in GBM. Here
we show that combining the CRAd Ad24-p53 with irradiation improves
the potency of both single treatments in vivo and in vivo. These results
show great promise for a future clinical trial combining radiotherapy with
Ad24-p53.
388. NEWCASTLE DISEASE VIRUS INDUCES APOPTOSIS IN
GLIOBLASTOMA CELLS
M. Remmers,
1
B. Knig,
2
R. Firsching,
1
and T.S. Schneider
1
;
1
2
Institute of Microbiology,
Otto-von-Guericke-Universitt Magdeburg, Magdeburg, Germany
Malignant gliomas remain incurable brain tumors despite aggressive
treatment with surgery, irradiation, and chemotherapy. As a new approach
the therapy with different kind of virus, for instance genetically engineered,
achieved more and more interest. Some naturally occurring strains of New-
castle disease virus (NDV) may have an oncolytic potential against tumor
cells. Therefore we asked whether NDV may induce cell death in glioblas-
toma cells. We analyzed the effects of lytic and non-lytic strains of NDV on
two established glioblastoma cell lines (U87, U138) with regard to parame-
ters of cell death. For this purpose the tumor cells were treated with NDV at
concentrations from 0.0001 to 100 HU (hemagglutination units) for up to
96 h. At different time points, cell viability was measured by using the tetra-
zolium salt WST-1. Additionally, the induction of apoptotic parameters was
investigated, as well as DNA-fragmentation with Tunnel-assay. Normal rat
astrocytes were treated as a control group. All three NDV strains induced
cell death in both glioblastoma cell lines in a dose-dependent manner. How-
ever, the most signicant effects were observed with the lytic NDV strain
73 T. In parallel we observed modulation of distinct key parameters in
the apoptotic signaling pathway, and especially DNA-fragmentation could
be detected as NDV-dose dependent. The same effects were not seen in
normal rat astrocytes. Oncolytic strains of NDV, described as apathogenic
for human, seem to be a very promising option for therapy of glioblastoma.
The in vitro results require an in vivo testing using an animal model as the
next step to therapeutical application.
389. GEMCITABINE SENSITIZES ASTROCYTOMA CELL
LINE TO RADIATION EXPOSURE INCREASING APOPTOSIS
A. Di Palma,
3
B. Bucci,
2
A. Citti,
2
E. Brunetti,
2
C. Carapella,
1
G. Raza,
3

R. Miceli,
3
and U. De Paula
3
;
1
Department of Neurosurgery, Regina
Elena Institute, Rome;
2
Research Center, Ospedale San Pietro AFaR-FBF
and
3
Oncological Radiotherapy Unit, Ospedale San Pietro FBF, Rome;
Italy
Gemcitabine (dFdCyd, Gem) is a deoxycytidine analogue, recently indi-
cated as a radiosensitizer agent in different tumor cell lines. In this study,
we investigated the ability of Gem to enhance the radiosensitivity of human
astrocytoma cell line (ADF). The effects of ionizing radiation (IR) and/or
Gem, on cell growth, cell cycle distribution, and apoptosis were assessed.
Cell counts and viability were determined by trypan blue exclusion test.
Cell cycle perturbation and apoptosis were analyzed by ow cytometry.
Escalating doses of 5, 10, and 15 Gy IR exposure caused a dose-dependent
inhibition on the cell proliferation. A 5-Gy IR exposure produced a moder-
ate antiproliferative effect, within 120 h from treatment, with an inhibi-
tory effect less than 30%. Conversely, 10-Gy IR treatment determined
signicant inhibition in cell growth (67%), already at 24 h after treatment
associated with an elevated amount of apoptosis (29%) at 48 h. Instead, in
cells treated with 15 Gy, the inhibition of cell growth was mainly due to
necrosis. For these experiments, we have chosen 10 Gy in combination with
Gem treatment (Gem/IR) at the IC
50
dose (0.0175 mM). The analysis of cell
growth showed that Gem/IR combined treatment enhances the lethal effect
induced by IR alone. Gem exposure, determining an accumulation in G
1

phase (about 64%) inuences cell sensitivity to IR-induced apoptosis. The
IR alone treatment induced a remarkable apoptotic cell death (40%) that
was increased by the Gem/IR exposure (68%). Gem sensitizes astrocytoma
cell line to the radiation exposure rendering these cells more prone to radia-
tion-induced apoptosis.
390. PRO-DRUG CONVERTING NEURAL STEM CELLS
FOR THE LOCAL INTRACEREBRAL CHEMOTHERAPY OF
HUMAN GLIOBLASTOMA XENOGRAFTS
N.O. Schmidt,
1
M. Ziu,
2
W. Przylecki,
2
T. Cargioli,
2
P. Black,
2
K.
Aboody,
3
and R. Carroll
2
;
1
2
Neurosurgical
Oncology Laboratory, Neurosurgery/Brigham & Womens Hospital,
Boston, Massachusetts, USA;
3
Divisions of Hematology/HCT, City of
Hope National Medical Center and Beckman Research Institute, Duarte,
USA
Previous reports have demonstrated that neural stem cells (NSCs)
distribute throughout experimental intracranial gliomas and are able to
track invading tumor cells when implanted in the adult rodent brain.
Based on this extensive tumor tropism NSCs are attractive candidates as
a potential delivery system for therapeutic gene products in the treatment
of invasive gliomas. Here, we investigated the therapeutic effectiveness of
glioma-targeting NSCs expressing cytosine deaminase (CD) to convert
systemically administered nontoxic pro-drug 5-uorocytosine (5-FC) to
5-uorouracil (5-FU), which diffuses out of the NSCs and selectively kills
dividing tumor cells. Murine NSCs C17.2 (lacZ/cytosine deaminase posi-
tive or negative) were stereotactically implanted distant (occipital) to well-
established intracerebral U87 human glioblastoma xenografts in adult nude
mice. Systemic treatment with 5-FC at 500 mg/kg/d started three days after
NSCs injection. Tumor growth was assessed by T1-Gd-enhanced magnetic
resonance imaging and NSCs distribution by X-gal immunohistochemistry.
Intracerebral implantation of 1.5 10
5
NSCs-CD followed by systemic
administration of 5-FC inhibited the tumor growth as assessed by MRI
14 days after treatment start. Furthermore, the survival was signicantly
prolonged when compared to the animals of the control groups (no NSC
implantation, implantation of NSC-mock plus 5-FC treatment or implanta-
tion of NSC-CD but no 5-FC). Histological analysis demonstrated the intra-
tumoral distribution of NSCs although the cells were initially implanted
distant from the main tumor mass. However, we were not able to detect
any NSCs based on X-gal immunohistochemistry in the brains of nude mice
analyzed later than 22 days after NSC injection. These results indicate that
NSCs represent a potent new delivery system for the local intracerebral
treatment of gliomas. However, NSCs may not survive within the tumor
environment for a prolonged time, and therefore larger numbers of NSCs or
multiple injections should be considered. Future studies need to address the
interaction of transplanted NSCs with the tumor environment.
391. THE EFFICACY OF ALGINATE ENCAPSULATED
CHO-K1 SINGLE CHAIN-TRAIL PRODUCER CELLS IN
THE TREATMENT OF BRAIN CANCER
J.M.A. Kuijlen,
1
B.J. de Haan,
2
J.J.A. Mooij,
1
W. Helfrich,
2
and P. de
Vos
1
;
1
Neurosurgery, Groningen;
2
Pathology and Laboratory Medicine,
Groningen University Hospital, Groningen; The Netherlands
Patients with astrocytic tumors in the central nervous system (CNS)
have low survival rates despite surgery and radiotherapy. Innovative thera-
pies and strategies must be developed to prolong survival of these patients.
The alginate microencapsulation method, used to continuously release a
certain cytotoxic drug in the vicinity of the tumor, is such a novel delivery
strategy. Targeted TRAIL (TNF-related apoptosis-inducing ligand) a death-
inducing protein with targeted specicity for a tumor-associated cell recep-
tor seems promising as an antineoplastic drug. TRAIL used in this study
was recombinantly coupled to a single chain variable fragment (scFv425)
with specicity for the EGF receptor. The biological functionality of the
apoptosis inducing scFv425:sTRAIL protein, which was released by the
microencapsulation method, was studied in vitro. Analysis of the intrace-
rebral biocompatibility of alginate capsules was performed by implantation
of empty alginate capsules in the brain of mice. Chinese hamster ovary cells
(CHO-K1) were recombinantly engineered to produce the single chain anti-
EGFR-TRAIL (scFv425:sTRAIL). The CHO-K1 producer cells were encap-
sulated in an alginate capsule with a semipermeable membrane through
which the scFv425:sTRAIL could be released. Empty alginate capsules were
implanted in the left cerebral hemisphere of C57BL6 mice. In vitro studies
show maintained biological functionality of the released scFv425:sTRAIL.
There was no tissue response detectable after intracerebral implantation of
the alginate capsules in C57BL6 mice brains. Biological functionality of the
produced scFv425:sTRAIL is maintained and intracerebral biocompatibility
of the capsules is warranted. Alginate encapsulation of CHO-K1scFv425:
sTRAIL producer cells and subsequently their intracerebral implantation is
technically feasible. This study justies further in vivo experiments.
392. MTOR AS A THERAPEUTIC TARGET FOR RADIATION
SENSITIZATION OF GLIOMA VASCULATURE
B. Lu; Vanderbilt University, Nashville, Tennessee, USA
It is known that radiation activates the PI3K/Akt pathway and that inhi-
bition of PI3K or Akt sensitizes tumor vasculature to radiotherapy. mTOR
is a downstream target of Akt, and we hypothesized that irradiation acti-
vates mTOR signaling in both glioma and endothelial cells. By inhibiting
this activation, we hypothesized that we could increase radiosensitization
of these cell lines. Two compounds which selectively inhibit mTOR, rapa-
mycin and RAD001 (everolimus), were used in this study. Both compounds
caused a signicant increase in sensitization of vascular endothelial cells
with only minor effects on glioma cell radiosensitivity as determined by
clonogenic assay. Therefore, we specically investigated the anti-angiogenic
effects of mTOR inhibitors. Increased phospho-mTOR protein was detected
in irradiated HUVEC cells with no detectable increase in total mTOR pro-
tein. Phospho-S6, a biomarker for mTOR signaling, was also found to be
induced following irradiation, and this effect was inhibited by PI3K or
mTOR inhibitors. Signicant increase in cleaved caspase 3 was detected
when Rad001 was combined with radiation. Endothelial tube formation
was signicantly diminished following treatment with rapamycin and 3 Gy.
Power-weighted Doppler of glioma xenografts in mice showed a signicant
reduction in vasculature and blood ow compared with mice treated with
3 Gy or RAD001 alone. We conclude that irradiation activates mTOR sig-
naling in vascular endothelium and that rapamycin and RAD001 increased
apoptosis of endothelial cells in response to radiation. To the authors best
knowledge, this is the rst study that demonstrates that mTOR inhibitors
may be a way to target the vasculature by radiosensitizing the vascular
endothelium resulting in better tumor control, as seen in experiments dem-
onstrating increased tumor growth delay in mice treated with rapamycin
with radiation compared with mice treat with either alone. We conclude
that mTOR inhibitors have increased efcacy as antiangiogenics when com-
bined with radiation.
393. HISTONE DEACETYLASE INHIBITOR VALPROIC
ACID EXTENDS SURVIVAL OF SCID MICE BEARING
ORTHOTOPICALLY HETEROTRANSPLANTED HUMAN
MEDULLOBLASTOMA CELLS THROUGH INDUCTION OF
APOPTOSIS
Q. Shu,
1
J. Su,
1
B. Antalffy,
2
S. Blaney,
1
C. Lau,
1
and X. Li
1
;
1
Pediatrics,
Texas Childrens Cancer Center;
2
Pathology, Baylor College of Medicine,
Houston, Texas, USA
Histone deacetylase (HDAC) inhibitors represent a novel family of
anticancer agents, several of which are being tested in clinical trials. Val-
proic acid (VPA), a widely utilized anticonvulsant with a well-established
toxicity prole, has recently been shown to inhibit HDAC. Our previous
studies demonstrated that VPA is capable of inducing apoptosis, cellular
differentiation, and senescence in vivo in medulloblastoma (MB) cell lines.
Additionally, VPA treatment also resulted in potent cell cycle arrest, pro-
liferation inhibition, and tumorigenicity suppression. These anti-tumor
activities were mediated through induction of histone (H3 and H4) acety-
lation and regulation of multiple gene expression. To evaluate VPAs anti-
MB effects in a preclinical model that closely recapitulates the biology of
human MBs, we injected a total of 10
5
cells from four MB cell lines (DAOY,
D283, MHH-MED-1, and MEB-MED-8A) into the right cerebellum of
Rag-2 SCID mice. Two weeks later, after formation of a tumor mass, treat-
ment with VPA through subcutaneously implanted osmotic pump (Aztec,
model 2001) was initiated. We were able to maintain serum VPA concen-
trations around 70 g/ml for 7 days, after which a new osmotic pump was
implanted to complete treatment for 2 weeks. Our results demonstrated
that VPA treatment prolonged the survival of tumorbearing animals for
all four cell lines. Accumulation of histone acetylation, increased apoptosis,
and induced cellular differentiation were observed in VPA-treated tumors.
Since the heterotransplanted MB cells proliferated in an anatomical site and
a microenviroment relevant to human MBs, our results offered compelling
preclinical evidence for testing VPA as an alternative or adjuvant therapy
for children with MBs. A clinical trial of VPA for children with recurrent
tumors has been initiated through the Childrens Oncology Group.
394. NON-PATHOGENIC POLIOVIRUS RECOMBINANTS
FOR THE TREATMENT OF MALIGNANT GLIOMA
M. Gromeier,
1
E.Y. Dobrikova,
1
M.K. Merrill,
1
S.A. Moore,
1
C. Kaiser,
1

R. Grisham,
1
and L. Selznick
2
;
1
Department of Molecular Genetics &
Microbiology and
2
Division of Neurosurgery, Duke University Medical
Center, Duke University Brain Tumor Center, Durham, North Carolina,
USA
We have developed a novel treatment modality for malignant glioma
based on genetically modied polioviruses. Susceptibility to poliovirus
depends on the availability of its cellular receptor, the immunoglobulin
superfamily molecule CD155. Selective expression of CD155 in spinal cord
motor neurons is believed to uniquely render this compartment susceptible
to poliovirus infection and ensuing destruction, producing the histopatho-
logical hallmarks of paralytic poliomyelitis. We have demonstrated that
the CD155 gene is upregulated ectopically in CNS malignancy, providing
a target for therapeutic intervention with poliovirus. However, the inher-
ent neuropathogenic potential of poliovirus would prohibit any therapeutic
application. We resolved this obstacle by genetic manipulation of a key viral
regulatory element involved in translation control. Poliovirus () strand
RNA, unlike cellular mRNA, is not equipped with a 5 terminal cap struc-
ture and hence uses an alternative mechanism for translation initiation.
A complex RNA structure within the 5 untranslated region of the viral
genome, the internal ribosomal entry site (IRES), mediates ribosomal entry
in a 5 end, cap-independent manner. We exchanged the IRES of poliovi-
rus with its counterpart from human rhinovirus type 2. Unexpectedly, the
resulting chimeric virus had completely lost the capacity to propagate in
normal neuronal cells. In a comprehensive study conducted at the FDA,
injection of the recombinant virus into the spinal cords of Cynomolgus
macaques failed to induce poliomyelitis. However, the chimera retained
wild-type growth potential and cell-killing ability in malignant glioma
cells. We have recently deciphered the molecular mechanisms that repress
rhinovirus IRES function in neuronal cells and favor viral propagation in
malignant glioma. Our observations demonstrate widespread perturba-
tions of the translation initiation apparatus in high-grade gliomas that
favor alternative mechanisms of protein synthesis at the IRES and promote
virus growth and cytotoxicity. A prototype oncolytic poliovirus/rhinovirus
recombinant has been produced for further safety evaluations at the FDA
and is scheduled to enter clinical investigation against glioblastoma multi-
forme in the near future.
395. THE SAFETY OF ADENOVIRAL VECTORS
CONTAINING THE HERPES SIMPLEX VIRUS THYMIDINE
KINASE GENE
G. Langford,
1
S. Yla-Herttuala,
2
and A. Boyd
1
;
1
Research and
Development, Ark Therapeutics, London, UK;
2
Gene Therapy Unit,
Kuopio University Hospital, Kuopio, Finland
Several clinical studies have demonstrated the safety and efcacy of
using adenoviral vectors containing the herpes simplex virus thymidine
kinase gene (Adv.HSV-tk) for the treatment of high-grade gliomas. To fur-
ther evaluate the safety of thymidine kinase in an adenoviral vector, a study
was performed to assess the immunological and toxicological responses to
intravenous or intracerebral administration of Adv.HSV-tk (Cerepro, Ark
Therapeutics Ltd). Ad.HSV-tk was administered to rats by intracerebral
or intravenous injection at doses of up to 1.2 1011 vps. The animals
were sacriced at days 3, 30, 60, 70, or 90 and the following assessments
were performed: macroscopic and microscopic analysis of all major organs,
assessment of clinical chemistry and hematological parameters, immuno-
logical analysis of inammatory reactions to Ad.HSV-tk, and PCR analysis
of the biodistribution of the viral vector. There was no effect on clinical
observations, body weight, food consumption, seminology, or clinical
parameters in either group. Minor changes in mean albumin/globulin lev-
els were observed in the intravenous groups compared to controls. At day
3, mean spleen weight was slightly increased, and myeloid hyperplasia was
observed in some animals receiving the highest dose of virus. At day 30 and
70 there was complete reversal of the splenic and bone marrow ndings.
Microscopic changes were seen at the site of injection of the control and
treated animals in the intracerebral groups. At day 3 inammatory cells
were seen at the meninges and at remote sites in a few animals. The extent
of the inltration was reduced at day 30, but changes were observed in the
neutrophil of some animals. PCR analysis showed that the amount of vector
decreased with time and was proportionally related to the dose level (from
day 3 to day 90). Vector was only detected at low levels in the urine at day
3. Intravenous or intracerebral administration of Ad.HSV-tk to rats is well
tolerated. Any microscopic observations were transient in the intravenous
group. The viral vector was cleared over time. Overall the results of this
toxicological study support the continued clinical use of Ad.HSV-tk.
396. INHIBITING PERIPHERAL BENZODIAZEPINE
RECEPTOR ENHANCES CHEMOTHERAPY-INDUCED
CYTOTOXICITY IN GLIOMAS
Y. Zhang,
1
S. Wu-Pong,
1
W. Broaddus,
2
and H. Fillmore
2
;
1
Pharmaceutics and
2
Neurosurgery, Virginia Commonwealth University,
Richmond, Virginia, USA
Therapeutic efcacy of adjuvant chemotherapy for the treatment of
glioblastomas remains poor in extending patient survival, in part due to
cellular resistance to apoptotic death. Peripheral benzodiazepine recep-
tor (PBR) is a putative member of the mitochondrial permeability transi-
tion pore which plays a critical role in controlling cellular apoptosis. PBR
expression is dramatically upregulated in brain tumors. We hypothesize
that overexpression of PBR may play a role in apoptotic resistance. Our
aims were to investigate the therapeutic effectiveness of inhibiting PBR
expression using antisense (AS) oligonucleotides (ODNs) in combination
with chemotherapy to enhance glioma cell death as well as to elucidate the
functional role of PBR in apoptosis in non-glioma cells. Two in vivo sys-
tems were established, both of which involve altering PBR expression levels
and comparing apoptotic susceptibility between control and mis-expressed
cells. In the glioma cell model, ve 18-mer phosphorothioate-modied AS
ODNs were designed. The most potent ODNs were selected based on their
ability to inhibit PBR protein expression in T98G human glioma cells using
Western blot analysis. Combination treatment of AS ODNs and camptoth-
ecin was assessed for changes in apoptosis (caspase-dependent PARP cleav-
age, TUNEL), and viability (trypan blue exclusion assay). In the lymphoma
cell model, an expression vector construct containing PBR cDNA was trans-
fected into Jurkat cells, a PBR-null cell line. Presence of PBR mRNA and
protein expression in stable transfectants was conrmed by using RT-PCR
and Western blot analysis, respectively. Extent of apoptosis (Annexin V
staining, TUNEL) was compared between PBR over-expressing clones and
vector control clones. T98G cells did not undergo any signicant extent of
apoptosis with camptothecin treatment alone. Of the ve AS ODNs, AS5
at 100 nM and 200 nM showed most potent inhibition (34 5% and 45
8%, P 0.05) of PBR protein expression. Combination treatment of AS
ODNs and camptothecin did not sensitize T98G cells to undergo apoptotic
death. However, enhanced cytotoxicity, in an AS ODN dose-dependent
manner, was observed. In Jurkat cells overexpressing PBR, apoptotic
response following camptothecin treatment was attenuated about 30% in
comparison to vector control cells. PBR protein expression was successfully
inhibited by AS ODNs. PBR downregulation via AS ODNs enhanced camp-
tothecin-induced cytotoxicity, possibly through apoptosis-independent
pathways in glioma cells. PBR overexpression in nonglioma cells partially
conferred resistance to apoptotic death, suggesting that targeting PBR pre-
sents an attractive therapeutic strategy to potentiate chemotherapy-induced
cytotoxicity in both gliomas and other types of cancer.
397. ALKYLGLYCEROL-MEDIATED INCREASE IN DRUG
DELIVERY TO THE NORMAL BRAIN AND TO BRAIN
TUMORS IN RATS: REGULATION OF DRUG TRANSFER
AND COMPARISON WITH HYPERTONIC MANNITOL AND
BRADYKININ
B. Erdlenbruch,
1
P. Hlper,
1
W. Kugler,
1
H. Eibl,
2
and M. Lakomek
1
;
1
Universittskinderklinik, Pdiatrie I, Gttingen;
2
Biophysikalische
Chemie, Max-Planck-Institut, Gttingen; Germany
Response of malignant brain tumors to chemotherapy is often poor or
transient due to poor penetration of most of anticancer agents across the
blood-brain barrier (BBB). The intracarotid administration of short-chain
alkylglycerols has been reported to be an effective and low toxic strategy
to increase the transfer of various cytotoxic drugs to the brain. We have
investigated the delivery of methotrexate (MTX) to the brain of normal
and of glioma-bearing rats (C6 and RG2) after i.a. injection of alkylg-
lycerols. Results were compared with those obtained after BBB-opening
using hypertonic mannitol or bradykinin. In tumor-free rats, alkylglycerols
induced a concentration-dependent increase in the delivery of MTX to the
brain. Using 1-O-pentylglycerol (120 and 150 mM) and 2-O-hexyldiglyc-
erol (75 and 100 mM), MTX-concentrations in the ipsilateral hemisphere
were increased 4- to 20-fold and 2- to 4-fold, as compared to controls.
Osmotic BBB disruption with 1.4 M mannitol resulted in a very strong
accumulation of MTX in the ipsilateral normal brain (114-fold), whereas
intracarotid infusion of bradykinin (10 g/kg/min) showed no effects in
normal animals. In glioma-bearing animals, 1.4 M mannitol increased
MTX concentrations 8.3-fold in the tumor and 15-fold in surrounding
brain. In contrast to this, a tumor to brain selectivity of the permeabiliz-
ing effect was observed using alkylglycerols and bradykinin; e.g., MTX
accumulation in C6 tumors amounted to 10.2 in tumor tissue and to 7.4
in surrounding brain after 2-O-hexyldiglycerol (100 mM). Bradykinin was
less effective, resulting in 2.4-fold higher MTX concentrations in tumor
tissue as compared to controls. Variations in the concentration and chemi-
cal structure of the alkylglycerols as well as in the time schedule of drug
administration were identied to be additional instruments to modify the
amount of drug delivered to the brain. From these data we conclude that
i.a. alkylglycerols are a promising new tool to circumvent the BBB in brain
tumor chemotherapy. This research was supported by Deutsche Krebshilfe
(10-1554-Er 2 and 10-1995-Er 3).
398. HYPOXIA-DRIVEN CD/5-FC TREATMENT
SUCCESSFULLY INITIATES BYSTANDER AND
RADIOSENSITIZATION EFFECTS IN A HYPOXIC
GLIOBLASTOMA CELL LINE
L. Hu, J. Chen, D. Wang, J. Wang, K. Lamborn, and D. Deen;
Department of Neurosurgery, University of California, San Francisco,
San Francisco, California, USA
Current therapy for glioblastoma relies on a multimodal approach.
However, efcacy of treatment is limited by therapeutic ratio: Doses suf-
cient to kill tumor cells often prove toxic to normal cells as well. Therefore,
it is especially important to develop therapies that are specically cytotoxic
to cancer cells, either directly or through bystander or sensitizing effects.
Hypoxic cells would be ideal targets for such an approach since they are spe-
cic to diseased tissue and often comprise the most treatment-resistant sub-
population of a tumor. Cytosine deaminase (CD) has been widely studied
as a form of suicide gene therapy, acting by deaminating the nontoxic pro-
drug 5-uorocytosine (5-FC) to form the highly cytotoxic 5-uorouracil
(5-FU). Previous studies have found that this strategy of treatment can
induce a bystander effect and radiosensitization in cancer cells. However,
none of these studies were conducted under hypoxic conditions, which are
prevalent in solid tumors and are typically resistant to therapeutic efforts.
Therefore, in this study, experiments were set to answer these critical
questions for our gene therapy strategy. We used a previously made gene
construct consisting of the SV40 minimal promoter under the control of
9 copies of hypoxia responsive elements (HRE). Under hypoxia, hypoxia
inducible factor-1 (HIF-1) becomes activated and binds to HRE sequences,
facilitating transcription of the yeast CD gene downstream. We performed
colony-forming efciency assays to assess survival of clonogenic cells, and
found that both a large bystander effect and radiosensitization occurred
under hypoxic conditions. This study was supported by CA-85356 and
NS-42927.
399. A NOVEL ULTRASONIC DEVICE FOR DISTRIBUTION
OF THERAPEUTIC PARTICLES: AN MRI-BASED
FEASIBILITY STUDY IN THE RAT BRAIN
Y. Mardor,
1
A. Kanner,
2
H. Ezer,
2
D. Daniels,
1
A. Ocherashvilli,
1
Y.
Wollman,
2
D. Massad,
3
M. Goldman,
3
and Z. Ram
2
;
1
Sheba Medical
Center-Tel Hashomer, The Advanced Technology Center, Ramat-Gan;
2
Neurosurgery Department, Tel-Aviv Medical Center, Tel-Aviv;
3
Sonenco
Ltd, Hedera; Israel
Ultrasonic Dispersion Device (UDD) is a novel tool for interstitial deliv-
ery of various therapeutic molecules and particles of various sizes. It is
composed of an ultrasound source that transmits ultrasound energy to a
probe with a drug-containing tip that is located within the tissue. Activa-
tion of the ultrasound (sonication) activates the spring-shaped tip and by
controlled vibration drives the particles into the tissue. The purpose of the
current study was to optimize UDD design and sonication parameters to
achieve a signicant homogenous distribution of particles in the normal rat
brain. MRI was used to assess the particle distribution in the brain immedi-
ately post-treatment and to follow up the particle concentration over time as
well as detect cytotoxic effects. The UDD was inserted stereotactically into
the rat brain under general anesthesia, with the active region located in the
center of the striatum. The probe was loaded with iron oxide (IO) nanopar-
ticles prior to and during treatment. Various UDD design parameters, such
as active region size and location along the probe, transmitter and cover
materials, and probe diameter and particle loading, as well as various ultra-
sonic transmission parameters, such as pulsation sequence and bandwidth,
were tested. Treatment duration was 1 to 5 min. Each set of parameters
was tested in 3 rats up to a total of 36 rats. Rats were scanned by gradi-
ent-echo and T1/T2-weighted MRI immediately post-treatment to assess
the IO distribution in the brain. Rats with homogenous IO distribution
were scanned periodically by MRI for up to 6 weeks to test the IO washout
timescale and possible cytotoxic effects. IO distribution in the rat brain
was obtained in 22 out of the 36 rats treated so far. The maximal diameter
of distribution, as calculated from the gradient-echo MR images, was 8
mm, and the maximal cross section of distribution was 0.42 cm
2
. Three
rats, in which a homogenous signicant IO distribution was observed,
were followed by MRI. In the rst follow-up scan, 4 days post-treatment,
a decrease (estimated 20%30%) in IO concentration was detected. The
remaining IO distribution was stable throughout the 6-week follow-up. No
cytotoxic effects were detected on the MR images. The preliminary data
demonstrates the unique advantages of the UDD as a minimal invasive tool
to achieve homogenous distributions of highly concentrated large particles
in brain tissue. Such distribution was achieved in extremely short treatment
durations. The long periods in which the large particles remain in the tissue
with no apparent toxic effects may enable distribution of large, slow-release
therapeutic agents coating the IO particles for effective treatment of CNS
pathologies.
400. UPTAKE AND DRUG DELIVERY USING
BIODEGRADABLE NANOPARTICLES IN MONOLAYERS AND
ORGANOTYPIC BRAIN SLICES
W. Meng,
1
T.L. Parker,
2
P. Kallinteri,
1
G.A. Hutcheon,
4
S. Higgins,
4
D.A.
Walker,
3
D.A. Walker,
3
and M.C. Garnett
1
;
1
School of Pharmacy and
2
School of Biomedical Science, University of Nottingham, Nottingham;
3
Queens Medical Centre, Nottingham;
4
School of Pharmacy and
Chemistry, Liverpool John Moores University, Liverpool; UK
The efcacy of brain tumor chemotherapy is limited by access of drug
to the brain. A therapeutic advantage may be achieved with biodegrad-
able nanoparticle (NP) drug delivery systems targeted to tumors. We aimed
to characterize the uptake and fate of NPs and the entrapped drug by a
medulloblastoma cell line (Daoy) and organotypic rat brain slice cultures.
For these in vivo studies we have used NPs prepared from a novel poly-
mer, which were stabilized by Tween-80, and contained the uorescent
dye rhodamine B isothiocyanate (RBITC) to represent the drug. NPs were
incubated with Daoy cells grown in monolayer culture for different incuba-
tion times or amounts of NPs. Intracellular uorescent dye was visualized
by uorescent microcopy and quantied by FACS. Cerebellar and cerebral
cortex rat brain slices were prepared from 2 day neonatal rats. After 15 days
of culture, RBITC-loaded NPs were added to the surface of slices and incu-
bated for 24 h. Quadruple uorescent labeling was used to co-label nuclei
(DAPI), to co-localize the NPs (RBITC) in the lysosomes (Lysotracker),
and to label macrophages (OX-42 monoclonal antibody) observed in the
confocal microscope. NPs were taken up by Daoy cells into lysosomes.
The uptake of NPs by Daoy cells was concentration and time dependent.
Intracellular uorescence intensity increased sharply within 2 h, and two
plateaus were established, one from 2 to 6 h and the other after 10 h of
incubation time. In organotypic slices, NP uptake was cell type dependent,
and few NPs were taken up by macrophages. Only a few NPs could be seen
in the extracellular space. Studying drug release from NPs, a reduction
of uorescence was observed using FACS. A time-series experiment using
uorescence microscopy showed about 50% of the dye had diffused out of
cells after 4h. These experiments aid our understanding of drug delivery by
nanoparticles. They show that few NPs were taken up by macrophages due
to Tween-80 stabilization and that NPs were taken up into the lysosomes,
rapidly degraded intracellularly, and showed release of both drug and NPs
into the culture medium in a few hours.
401. DEVELOPMENT OF TRAIL THERAPEUTIC
STRATEGIES FOR MALIGNANT GLIOMAS
J.H. Song,
1
D. Song,
1
C. Lin,
2
S. Phuphanich,
1
D. Brat,
1
J. Olson,
1
E. Van
Meir,
1
and C. Hao
1
;
1
Pathology & Laboratory Medicine, Neurosurgery,
Hematology & Oncology, Emory University, Atlanta, Georgia, USA;
2
Medical Research, China Medical University Hospital, Taichung,
Taiwan
Tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL)
induces apoptosis in malignant glioma cells and thus offers a new poten-
tial therapeutic agent for malignant gliomas. Unfortunately, many human
malignant glioma cells are resistant to TRAIL, posing a major obstacle in
TRAIL therapy. In this study, we investigated inhibitory mechanisms for
development of combination treatments that can overcome the resistance.
First, we examined a large panel of 26 human malignant glioma cell lines
for their sensitivity to TRAIL and grouped them into two phenotypes,
TRAIL sensitive and resistant. The resistant cell lines were analyzed for
the sensitivity to the combination treatment with TRAIL and chemotherapy
drug cisplatin and etoposide and divided into two subtypes, chemotherapy-
sensitized resistant and complete resistant. TRAIL induced apoptosis
through binding of cell surface death receptor DR4/DR5, leading to the
assembly of death-inducing signaling complex (DISC) in the sensitive cells. In
the DISC, apoptosis-initiating caspase-8 was cleaved and thus initiated cas-
pase cascade leading to programmed cell death (apoptosis). In contrast, cel-
lular Fas-associate death domain-like, IL-1b-converting enzyme-inhibitory
protein (c-FLIP) and phosphoprotein enriched in diabetes (PED) were
recruited to TRAIL-induced DISC and inhibited caspase-8 cleavage in the
chemotherapy-sensitized resistant cells. Targeting c-FLIP and PED with
small interfering RNA (siRNA) and the chemotherapy drugs sensitized the
cells to TRAIL-induced apoptosis. The complete resistant cell lines were
analyzed by the combined comparative genomic hybridization (CGH),
G-banding/spectral karyotyping (SKY), and uorescence in situ hybrid-
ization (FISH) analyses with chromosomal region specic probes used to
identify aberration of chromosomal regions that harbor key TRAIL sig-
naling gene DR4/DR5, caspase-8, capsase-3, caspase-7, caspase-9, Bid,
Bax, Bak, Bcl-2, and Smac. Loss or structural aberration of chromosome
regions harboring DR4/DR5, caspase-8, Bid and Smac was simultaneously
detected in the complete resistant but not sensitive cell lines. In conclusion,
this study provides new TRAIL combination therapeutic strategies that tar-
get the DISC resistance. In addition, this study identies genetic markers
that could be used to predict the responsiveness of malignant glioma to the
TRAIL-based therapies.
402. THE INHIBITION OF THROMBOXANE SYNTHASE
ACTIVITY: A NOVEL TARGET FOR THE TREATMENT OF
MALIGNANT GLIOMAS
N.O. Schmidt,
1
M. Ziu,
2
T. Cargioli,
2
A. Giese,
3
M. Westphal,
1
P.
Black,
2
and R. Carroll
2
;
1
Neurosurgery, Universittsklinikum Hamburg-
Eppendorf, Hamburg, Germany;
2
Neurosurgical Oncology Laboratory,
Neurosurgery, Brigham & Womens Hospital, Boston, Massachusetts,
USA;
3
Department of Neurosurgery, University Schleswig-Holstein,
Luebeck, Germany
Thromboxane synthase (TXSA), an enzyme of the arachidonic acid
metabolism, is upregulated in human glial tumors and involved in the
regulation of glioma cell invasion, which is one of the major limiting fac-
tors of current therapeutic strategies in this disease. Recent in vitro stud-
ies indicate that the inhibition of thromboxane synthase activity at non-
cytotoxic concentrations blocks the invasive phenotype of glioma cells. This
in turn increases a pro-apoptotic disposition and therefore the susceptibility
to standard apoptosis-inducing chemotherapeutic compounds like BCNU,
camptothecin, and etoposide. Here, we evaluated the therapeutic in vivo
effects of furegrelate, a clinically tested TXSA inhibitor, in orthotopic U87
human glioblastoma xenografts by using local intracerebral microinfusions
at 0.5 and 2 mg/kg/d. Local delivery of furegrelate by osmotic mini-pumps at
2 mg/kg/d for 21 days signicantly inhibited the growth of well-established
orthotopic gliomas in a nude mice model (76.12%, P 0.005). Furegrelate
and BCNU displayed strong synergistic effects on the in vitro induction of
U87 glioma cell apoptosis as measured by an ELISA for DNA fragmenta-
tion. Therefore, we assessed the effects of furegrelate alone and in combina-
tion with BCNU on the survival of human glioma bearing nude mice. While
local delivery of furegrelate alone at 2 mg/kg/d for 28 days prolonged the
survival only marginally, the combination with systemically administered
BCNU (15 mg/kg/d) enhanced the survival more than the single compounds
alone. Our results indicate that targeting of the increased TXSA activity
in human gliomas inhibits tumor growth in vivo by inducing pro-apop-
totic, anti-proliferative, and anti-angiogenic effects. Local treatment with a
TXSA inhibitor has the potential to enhance conventional chemotherapeu-
tic schemes for malignant gliomas. Future studies need to evaluate modern
modulators of TXSA activity and address their effects in relationship to
other metabolites of the arachidonic acid pathway.
403. INHIBITION OF 90-KD HEAT SHOCK PROTEIN
POTENTIATES THE CYTOTOXICITY OF DNA-
ALKYLATING AGENTS IN HUMAN GLIOMA CELLS
S. Ohba, Y. Hirose, and T. Kawase; Neurosurgery, Keio University,
Tokyo, Japan
Since gliomas are not curable surgically, it is necessary to develop an
effective regime of adjuvant therapies, such as radio- or chemotherapy.
Several ways to potentiate the cytotoxicity of antitumor agents have been
reported. These include cell-cycle checkpoint abrogation, depression of
anti-apoptotic protein expression, and depletion of DNA repair enzymes.
Thus, many pathways can be targeted in an effort to sensitize tumor cells to
chemotherapeutic agents. Previous studies revealed that a molecular chap-
erone 90-kD heat shock protein (Hsp90) is expressed at higher levels in
human neoplastic tissues, including gliomas, than in normal tissues. Hsp90
participates in the stability and functions of its client proteins, which are
involved in cell-cycle regulation (ex. Wee1, Plk1), cell survival (ex. Akt,
survivin), and oncogenesis (ex. raf-1, src), and it is involved in a cytopro-
tective mechanism against cellular stresses, such as DNA damage. We
hypothesized that Hsp90 inhibitors might act as antitumor agents against
gliomas and potentiate the cytotoxicity of DNA-damaging agents. In the
present study, we found that at a low concentration (3 nM) the Hsp90
inhibitor geldanamycin (GA), an ansamycin derivative, reduced the clono-
genicity of U87MG human glioma cells in a p53-independent manner, and
that GA potentiated the cytotoxicity of DNA-alkylating agents temozolo-
mide and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) on human glioma
cells at a lower concentration (1 nM). This GA-induced potentiation of
DNA-alkylating agent-induced cytotoxicity was not a consequence of G
2

checkpoint abrogation or degradation of the anti-apoptosis proteins Akt or
survivin, and exogeneous Akt overactivation did not overcome GA-induced
sensitization of U87MG cells to DNA-alkylating agents. Experiments using
another Hsp90 inhibitor, radicicol, a macrocyclic antibiotic, showed the
results similar to those mentioned above. Although the mechanism of the
GA-induced enhancement of the cytotoxicity of DNA-alkylating agents is
still unclear, since two different types of Hsp90 inhibitors showed poten-
tiation of the cytotoxicity of DNA-alkylating agents, and since this effect
of Hsp90 inhibitors was clearly recognized with very low concentration of
compounds that can be toxic to normal cells at much higher concentration,
we conclude that Hsp90-targeted therapy may provide an effective strategy
for the chemosensitization of human gliomas.
404. ADULT HUMAN MESENCHYMAL STEM CELLS: VEGF-
DRIVEN INTERACTION WITH GLIOMA CELLS IN VITRO
C. Schichor,
1
N. Etminan,
1
S. Miebach,
1
S. Grau,
1
T. Birnbaum,
2
C.
Padovan,
2
J. Tonn,
1
and R. Goldbrunner
1
;
1
Neurosurgery;
2
Neurology,
Clinic Univers. LMU Munich Grosshadern, Munich, Germany
Much effort has been put into establishing human multipotent cells as car-
riers for malignant glioma therapy. The aim of our study were (1) to character-
ize factors that inuence active movement of stem cells in the environment of
a tumor inltrated brain and (2) to test human adult mesenchymal stem cells
(MSCs), which are easily available through bone marrow biopsy, for their
migratory and invasive behavior and their interaction with human gliomas.
Human MSC were isolated from bone marrow biopsies carried out for
hematological indications. Migration of human adult MSC- and rodent
embryonal NSC-spheroids (cell line C17.2) was studied on different matri-
ces: laminin, tenascin, and plastic. Tumor-conditioned medium as well as
VEGF were added in order to evaluate the role of glioma derived factors. To
assess invasion, confrontational co-cultures of glioma (U373 GFP, C6 GFP,
C6 VEGF sense, C6 VEGF antisense transfected) and stem cell spheroids
(human MSC and rodent NSC, respectively) were investigated. Invasion
was visualized by light and confocal microscopy. Migration of both rodent
embryonal NSC and human adult MSC was fastest on laminin, when com-
pared to tenascin and plastic. VEGF as well as tumor-conditioned medium
signicantly increased NSC and MSC migration. Human MSCs showed
an extensive invasion into glioma spheroids, even more than embryonal
rodent NSCs. Invasion of NSC into VEGF sense C6 spheroids was much
more rapid than invasion into VEGF antisense spheroids. Both NSC and
MSC show intensive migratory behavior in the presence of glioma cells
and glioma-conditioned medium. Obviously, VEGF is a crucial factor in
enhancing stem cell motility.
405. IN VITRO SAFETY AND EFFICACY OF A NOVEL CB2-
SELECTIVE CANNABINOID CHEMOTHERAPEUTIC AGENT,
KM-233, FOR THE TREATMENT OF HIGH- GRADE GLIOMA
C. Duntsch,
1
M. Divi,
1
T. Jones,
1
G. Wood,
1
B. Moore,
1
and A. Sills
1
;
1
Neurosurgery and
2
Pharmaceutical Sciences, University of Tennessee,
Memphis, Memphis, Tennessee, USA
Currently accepted therapeutic adjuvants to surgery for the treatment
of high-grade gliomas such as radiotherapy and chemotherapy provide only
a minor improvement in the disease course and life expectancy. Recently,
it has been demonstrated that ligands of the CB1/CB2 receptors have vary-
ing degrees of cytoxicity against a variety of cancer cell lines. We have
completed in vitro studies designed to test the efcacy and safety of a novel
chemotherapeutic agent, KM-233, for the treatment of high-grade glioma.
KM-233 is a classical cannabinoid with good blood-brain barrier penetra-
tion that possesses a 2.3- and 27-fold higher afnity for the CB-1 and CB-2
receptors, respectively, relative to THC. In vitro tissue culture cytotoxic-
ity assays were used to measure the anti-tumor effects of KM-233 against
human U87 glioma cells. KM-233 was found to have signicant cytotoxic
effects against U87 human glioma cells and reproducibly demonstrated a
fty-percent inhibitory concentration (IC50) of 1.42 mM. Similar assays
were used to compare the cytotoxic efcacy of KM-233 to
8
-tetrahydro-
cannabinol and BCNU. In these studies, KM-233 was as efcacious in its
cytotoxicity as
8
-tetrahydrocannabinol, and far superior to the commonly
used anti-glioma chemotherapeutic agent BCNU. Kinetic studies of the
onset of activity of KM-233 demonstrated that cytotoxic effects of KM-
233 against human glioma cells in vitro occur as early as two hours after
administration. Furthermore, we found that the dosing of KM-233 can be
cycled on a daily basis without compromising its cytotoxic efcacy, thereby
limiting potential toxicity from excessive exposure in vivo. To test the safety
and toxicity of KM-233 against healthy adult brain tissue, an organotypic
brain slice coculture model of cortex, striatum, and substantia nigra was
used for dose-escalation studies. We found that, while there is some mini-
mal toxicity associated with continuous administration of KM-233 in an
organotypic brain slice culture model, cycling of KM-233 at doses that are
exquisitely cytotoxic to glioma cells were well tolerated by healthy cultured
brain tissue. These studies provide in vitro evidence that KM-233 shows
promising efcacy against cultured glioma cell lines, shows minimal toxic-
ity to healthy cultured brain tissue, and should be considered for preclinical
development in animal models of glioma.
406. CONVECTION-ENHANCED DELIVERY OF LIPOSOMAL
DOXORUBICIN ERADICATES U251MG INTRACRANIAL
BRAIN TUMORS IN RATS
R. Saito,
1
M. Krauze,
2
C. Noble,
3
T. Kumabe,
1
T. Tominaga,
1
J. Park,
3

M. Berger,
2
and K. Bankiewicz
2
;
1
Neurosurgery, Tohoku University,
Sendai, Japan;
2
Neurological Surgery and
3
Hematology-Oncology,
University of California San Francisco, San Francisco, California, USA
Convection-enhanced delivery (CED) of liposomes into brain and brain
tumor xenograft models resulted in robust tissue distribution and could be
detected by MRI (Saito, Cancer Res., 2004). Image-guided CED of thera-
peutic liposomes for treatment of brain tumors is a lofty goal. The efcacy
of well-characterized clinically available liposomal doxorubicin liposomal
drug delivered by CED was evaluated in U251MG human glioblastoma
intracranial xenografts. CED of liposomal doxorubicin at a dose safe to
the normal brain (0.2 mg/ml doxorubicin) was signicantly more effective
than systemic administration at the maximum tolerable dose. When CED of
liposomal doxorubicin was compared with free drug, liposomal drug dem-
onstrated a higher therapeutic index, resulting from improvements in both
efcacy and safety. When used at a nontoxic dose, liposomal doxorubicin
demonstrated improved survival over free doxorubicin at the same dose. In
addition, at a tenfold higher dose (2 mg/ml doxorubicin), liposomal doxo-
rubicin was less toxic than free doxorubicin. A study of the tissue distribu-
tion following CED revealed liposomal doxorubicin distributed over larger
regions in the brain parenchyma and resulted in longer tissue retention of
the drug at the site of initial distribution. Free drug, when infused by CED,
did not distribute as well as liposomal doxorubicin and induced early onset
tissue damage, which led to increased tissue toxicity. CED of liposomal
doxorubicin shows promise for treating brain tumors. The combination
with imaging may provide an effective strategy for brain tumor therapy.
407. SYNERGISTIC INTERACTION BETWEEN 17-AAG AND
PHOSPHATIDYLINOSITOL 3-KINASE INHIBITION IN
HUMAN MALIGNANT GLIOMA CELLS
D. Premkumar, B. Arnold, A. Engram, and I. Pollack; Neurosurgery,
Childrens Hospital of Pittsburgh, Pittsburgh, Pennsylvania, USA
The PI3K/Akt pathway is often constitutively activated in malignant
glioma cells, in many cases as a result of mutation of PTEN, an endog-
enous inhibitor of Akt, which renders tumor cells resistant to cytotoxic
insults, including those related to anticancer drugs. Pharmacological inhi-
bition of this pathway may potentially restore or augment the effectiveness
of conventional chemotherapy or other signaling-targeted agents. Because
the heat shock protein (HSP) is involved in the conformational maturation
of a number of signaling proteins critical to the proliferation of malignant
glioma cells, we hypothesized that the combination of the PI3K inhibitor
LY294002 and the HSP90 inhibitor 17-AAG would promote glioma cyto-
toxicity by decreasing both the activation status and levels of Akt, as well as
downregulating the levels of other relevant signaling effectors. We therefore
examined the effects of the LY294002 and 17-AAG, alone and in combina-
tion, on signal transduction and apoptosis in a series of malignant glioma
cell lines. Simultaneous exposure to these inhibitors signicantly induced
cell death and irreversibly inhibited proliferative activity and colony-forming
ability of the glioma cell lines. Quantitative analysis revealed that enhance-
ment by LY294002 of 17-AAG-induced cytotoxicity was synergistic, lead-
ing to a pronounced increase in active caspase-3 and PARP cleavage. No
signicant growth inhibition or caspase activation was seen in control cells.
The enhanced cytotoxicity of this combination was associated with dimin-
ished Akt activation and a signicant downregulation of epidermal growth
factor receptor (EGFR), Raf-1, and mitogen-activated protein kinase
(MAPK). Combination of 17-AAG and LY294002 did not modify phos-
pho JNK/SPK, or p38 MAPK. Cells exposed to 17-AAG and LY294002
displayed a signicant reduction in cell cycle regulatory proteins, such as
pRb, CDK4, CDK6, and cyclin D1. Taken together, these ndings suggest
that the PI3K/Akt pathway plays a critical role in regulating the apoptotic
response to 17-AAG and that targeting this pathway could provide a potent
strategy to treat patients with malignant gliomas.
408. DOWNMODULATION OF E1A PROTEIN EXPRESSION
AS A NOVEL STRATEGY TO DESIGN CANCER SELECTIVE
ADENOVIRUSES
H. Jiang,
1
D. Medrano,
1
C. Gomez-Manzano,
1
M. LeMoine,
1
R.
Alemany,
2
M. Olson,
1
M. Alonso,
1
C. Conrad,
1
W. Yung,
1
and J. Fueyo
1
;
1
Neuro-Oncology, The University of Texas M.D. Anderson Cancer
Center, Houston, Texas, USA;
2
Institut Catala dOncologia, Barcelona,
Spain
Oncolytic adenoviruses are being tested as potential therapies for
human malignant tumors, including gliomas. Here we report for the rst
time that the deletion of a 48-60 aa region in the CR1 domain of E1A
resulted in low levels of E1A protein, conditioning the replication of the
mutant adenoviruses specically to cancer cells. In this study, we com-
pared the oncolytic potencies of three mutant adenoviruses encompassing
deletions within the CR1 (Delta-39) or CR2 (Delta-24) regions or in both
regions (Delta-24/39) of E1A protein. Analyses of cell viability showed a
comparable cytopathic effect of Delta-24 and Delta-39, and viral replica-
tion studies revealed similar replication capability for both adenoviral con-
structs in glioma cells, although Delta-24/39 displayed more attenuated
potency. Importantly, the activity of Delta-39 was signicantly attenuated
compared to Delta-24 in proliferating normal human astrocytes. Direct
analyses of the activation of E2F-1 promoter demonstrated the inability of
Delta-39 to induce S-phase-related transcriptional activity in normal cells.
Interestingly, immunoblotting analysis showed that E1A protein levels in
cells infected with Delta-39 were remarkably downmodulated. Further,
protein stability studies through inhibiting protein synthesis with anisomy-
cin revealed enhanced degradation of CR1-mutant E1A proteins, and block
of the proteasome activity with lactacystin resulted in the striking rescue of
the E1A levels. Collectively, our data showed that, compared to Delta-24,
the deletion in Delta-39 resulted in suboptimal expression level of E1A pro-
tein, leading to signicant attenuation of the virus in normal astrocytes but
still maintaining efcient replication in glioma cells. We conclude that the
level of E1A protein is a critical determinant of the selectivity of oncolytic
adenoviruses, and we propose a completely novel strategy for the design and
construction of conditionally replicative adenovirus.
409. ENHANCED CELLULAR RETENTION OF AN
INTERNALIZING ANTI-EGFRVIII MONOCLONAL
ANTIBODY RADIOIODINATED USING LYS5-
[*I]IODOBENZOYL GLY1-MALEIMIDO GEEEK ([*I]IB-
MAL-D-GEEEK), A PROSTHETIC GROUP CONTAINING
NEGATIVELY CHARGED D- GLUTAMATES
G. Vaidyanathan, K.L. Alston, P.C. Welsh, and M.R. Zalutsky;
Radiology, Duke University Medical Center, Durham, North Carolina,
USA
Monoclonal antibodies (mAbs) reactive to EGFRvIII, a mutant form of
the epidermal growth factor receptor expressed by gliomas but not by nor-
mal tissues, are rapidly internalized and degraded after binding to EGFR-
vIII-expressing cells. If mAbs are radioiodinated directly on the tyrosine
residues, the radiolabeled catabolite iodo-tyrosine is rapidly washed out of
the tumor. Therefore, if anti-EGFRvIII mAbs are to be useful in radioimmu-
notherapy, radiohalogenation methods with which enhanced tumor reten-
tion of radioactivity can be achieved are necessary. Earlier, we evaluated a
radioiodinating agent [
*
I]D-KRYRR containing a tyrosine for labeling, 3
arginines for positive charge, and a lysine for mAb coupling via a maleimido
bifunctional agent. In this study, we evaluated a novel agent with 3 nega-
tively charged D-glutamates for lysosomal trapping, an iodobenzoyl moiety
for minimizing dehalogenation, and a maleimido group for conjugation to
mAb. Maleimidoglycine was prepared as a precursor for Gly
1
-maleimido
GEEEK preparation by solid-phase peptide synthesis. The lysine side chain
of Gly
1
-maleimido GEEEK was derivatized with 3-iodobenzoyl and 3-(tri-
n-butylstannyl)benzoyl moieties by treatment with the respective N-succin-
imidyl benzoyl esters to obtain the iodo standard and tin precursor, respec-
tively. Radioiodination of the tin derivative yielded [
125
I]IB-Mal-D-GEEEK
in 90.3 3.9% radiochemical yields. This radioiodinated agent was con-
jugated to iminothiolane-treated L8A4, an anti-EGFRvIII mAb in 54.3
17.7% conjugation yields. The protein-associated radioactivity (methanol
precipitation) of the labeled mAb was 94.3 5.8%, and the immunore-
active fraction was 82.3 2.2% (Lindmo method). In vitro assays with
the U87EGFR glioma cell line indicated that internalized radioactivity
for [
125
I]IB-Mal-D-GEEEK-L8A4 conjugate increased from 14% at 1 h to
45% at 24 h. On the other hand, from a paired-label study, only 7% of the
radioactivity from the directly radioiodinated L8A4 was internalized at 1
h, and the value steadily decreased, reaching 3% at 24 h. In comparison,
internalized radioactivity for L8A4 radioiodinated with [
*
I]D-KRYRR in
the same cell line decreased from about 40% at 2 h to about 20% at 24 h.
These results suggest that [
125
I]IB-Mal-D-GEEEK is a promising reagent
for the radioiodination of internalizing mAbs. We are currently evaluating
L8A4 radioiodinated using this new method in EGFRvIII-expressing tumor
xenograft models.
410. S-FARNESYLTHIOSALICYLIC ACID TARGETS RAS
SIGNALING IN A MOUSE MODEL OF GLIOMA
Y. Lyustikman,
1
Y. Kloog,
3
and E. Holland
1,2
;
1
Cancer Biology and
Genetics,
2
Surgery (Neurosurgery), Memorial Sloan-Kettering Cancer
Center, New York, New York, USA;
3
Neurochemistry, Tel Aviv
University, Tel Aviv, Israel
The Ras signal transduction cascade is commonly activated in high-
grade gliomas. Evidence suggests that farnesylation is critical for recruit-
ment of Ras to the membrane and its subsequent activation. However,
treatment with several farnesyltransferase inhibitors (FTIs) has failed to
achieve therapeutic benet. This failure may result from resistance by evolv-
ing alternative prenylation mechanisms. By contrast, S-farnesylthiosalicylic
acid (FTS) is a competitive inhibitor of Ras that likely acts by displacing
Ras from the plasma membrane and that does not require inhibition of
farnesylation for its effect. Therefore, it may be more effective than FTI
therapy. We explored the mechanism of action of FTS in vitro and the thera-
peutic efcacy of FTS on a mouse model of glioma in vivo. We transformed
neural progenitor cells with activated forms of Kras, Akt, Kras Akt, or
PDGF-B by somatic gene transfer. Transformed progenitors were exposed
to increasing doses of FTS and analyzed for changes in morphology, induc-
tion of apoptotic cell death by ow cytometry, and signaling effects by
Western blotting. Synergistic effects with other drugs were addressed by
combined treatments with a MEK inhibitor, an mTOR inhibitor, or an
Akt inhibitor. Subcellular localization of Kras was determined by immu-
nouorescence microscopy. Finally, we assessed the potential therapeutic
effects of FTS in vivo in a Kras-driven glioma model. FTS induced rapid
onset of apoptosis in a dose-dependent and cell linedependent manner.
Specically, at low concentrations of the drug, only the Ras transformed
cells underwent apoptosis, whereas at high doses FTS exhibited a general-
ized toxicity toward all cell lines. This suggests specicity for FTS activity
against Ras at low concentrations. Addition of constitutively active Akt
protected against FTS-induced apoptosis at lower doses, and combined
treatment with an Akt inhibitor restored sensitivity. The rescue provided
by Akt was mTOR independent because addition of an mTOR inhibitor had
no effect. Experiments are underway to examine the effects of FTS in vivo.
FTS induces apoptosis in cells that are reliant on Ras signaling to maintain
their transformed characteristics in a dose-dependent manner. Activation
of Akt provides a rescuing effect that is abolished by treatment with an
Akt inhibitor. These results suggest that FTS may potentially be a novel
therapeutic agent for treatment of tumors with increased Ras signaling. As
activation of Akt is also common in high-grade gliomas, combined therapy
with FTS and an Akt inhibitor may be clinically useful and more effective
than with either drug alone.
411. SYNERGISTIC AUGMENTATION OF VINCRISTINE-
INDUCED CYTOTOXICITY BY PHOSPHATIDYLINOSITOL
3-KINASE INHIBITOR IN HUMAN MALIGNANT GLIOMA
CELLS: EVIDENCE FOR THE INVOLVEMENT OF P38 AND
ERK SIGNALING PATHWAY
I. Pollack, D. Premkumar, J. Mathas, and B. Arnold; Neurosurgery,
University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania, USA
Microtubule-interfering agents (MIAs), such as vincristine, are widely
used for the treatment of cancer, and are included in many treatment
regimens for childhood brain tumors. The anticancer properties of MIAs
have been attributed in part to interference with microtubule assembly,
impairment of mitosis, and cytoskeletal changes, with additional effects on
mitogen-activated protein kinase signaling and caspase activation. Because
malignant gliomas commonly have dysregulation of PI3K/Akt signaling,
which can promote cell survival and potentially limit the activity of such
agents, we questioned whether PI3K inhibition with LY294002 could
potentiate the efcacy of vincristine in a panel of glioma cell lines versus
normal astrocytes. We therefore examined the effects of the LY294002 and
vincristine, alone and in combination, on cell survival, signal transduction,
and apoptosis in a series of malignant glioma cell lines versus normal astro-
cytes. Simultaneous exposure to these inhibitors signicantly induced cell
death and inhibited proliferation and clonogenicity of the glioma cell lines.
Quantitative analysis revealed that enhancement by LY294002 of vincris-
tine-induced cytotoxicity was synergistic, leading to pronounced caspase
activation at concentrations that had no signicant effects on control cells.
The enhanced cytotoxicity of this combination was associated with signi-
cant activation of p38 MAPK signaling and induction of G2/M blockade on
cell cycle analysis. Pretreatment with either SB203580 or z-VAD.fmk, selec-
tive inhibitors of p38 MAPK and caspase signaling, respectively, abrogated
the apoptotic response to the combination of LY294002 and vincristine.
Taken together, these ndings demonstrate that PI3K/Akt inhibition can
potentiate the effects of vincristine and that the combination of molecularly
targeted therapies and conventional agents could provide a potent strategy
to treat patients with malignant gliomas.
412. COMBINATION OF AN ANGIOGENESIS INHIBITOR
WITH RADIOTHERAPY FAILED TO SHOW SYNERGISM IN
AN ORTHOTOPIC MURINE GBM MODEL
J. Verhoeff,
1
L. Stalpers,
2
J. Haveman,
2
D. Troost,
3
M. Ramkema,
3

D. Bosch,
1
and W. van Furth
1
;
1
Neurosurgery,
2
Radiation Therapy,
and
3
Neuropathology, Academic Medical Centre, Amsterdam, The
Netherlands
Clinical effects of irradiation in GBM patients are likely the result of
a direct anti-neoplastic effect, as well as radiation-induced damage to the
tumor vasculature. We hypothesized that additional damage to the tumor
vessels, by combining radiotherapy with an angiogenesis-inhibitor, will
enhance radiotherapy effectiveness. An orthotopic murine GBM (U251-
NG2) model was used, which we modied to allow focal brain irradiation.
The antibody against murine VEGF-R2 (DC101) was given in a dose of
40 mg/kg every 3 days, starting on the same day as the irradiation (2 mCi
iodine seeds). Treatment started 7 days after stereotactic injection of the
cells in the right frontal lobe. Both treatment modalities were controlled
with sham treatment, resulting in 4 groups. Mice were sacriced when los-
ing 20% weight, showing neurological signs, or after 13 weeks after cell
inoculation. Tumor take in 96 athymic nude mice was 90%. Control mice,
getting only sham treatments, had a mean survival of 5.9 weeks. Focal brain
irradiation improved mouse survival statistical signicantly, with a median
survival of 9.9 weeks. DC101 did not affect the median survival (5.1 weeks),
although histological analysis did show a changed vascular pattern in these
tumors. By combining DC101 with focal brain irradiation the favorable
effects of irradiation on survival were lost, with a median survival of 5.9
weeks. In our orthotopic murine GBM model we could not nd a coop-
erative effect of DC101 with radiotherapy. In fact, a contrary effect was
shown, with a survival similar to control groups. Histology showed altered
tumor vasculature in the mice that received DC101, but no explanation for
the worsened outcome of the combination therapy. We suggest that receptor
blockage may have resulted in increased local VEGF concentrations and
physiological effects through the VEGF-R1. New experiments will be con-
ducted to explore the result of combining radiation therapy with a therapy
that directly binds to the VEGF molecule.
413. EFFICACY AND SAFETY OF A REPLICATION-
RESTRICTED VSV FOR THE TREATMENT OF
GLIOBLASTOMA USING ORGANOTYPIC BRAIN SLICE AND
RODENT MODELS OF INTRACRANIAL GLIOMA
A. Sills,
1
C. Duntsch,
1
H. Jayakar,
4
Q. Zhou,
1
Z. Xiang,
1
L. Pfeffer,
2
J.
Robertson,
1
and M. Whitt
3
;
1
Neurosurgery,
2
Pathology and Laboratory
Medicine, and
3
Molecular Sciences, University of Tennessee at Memphis,
Memphis, Tennessee;
4
GTX, Inc., Memphis, Tennessee; USA
Vesicular stomatitis virus (VSV) is an enveloped negative strand RNA
virus being evaluated for use in treatment a variety of tumors. A sensi-
tive organotypic brain tissue slice-glioma coculture system was used to
evaluate use of recombinant, wild-type VSV (wt-VSV) in the treatment of
glioma. Previous work has shown that even when replication of wt-VSV was
blocked by pretreatment of the slice culture with interferon-b, the integrity
of neuronal tissues were signicantly damaged following exposure to wt-
VSV. This neurotoxicity would pose a major limitation for clinical use of
wt-VSV. In this report, we describe the use of a recombinant, replication-
restricted second-generation VSV, GTx-v401, as a potential candidate for
glioma therapy. In contrast to the results observed with wt-VSV, we found
that GTx-v401 exhibited minimal cytotoxicity in the organotypic slice cul-
ture while displaying high levels of oncolytic activity toward glioma cells
growing within the slice culture. We also observed no virus-induced loss
of neuronal integrity as measured by MAP-2 staining and no change in the
electrophysiological properties of the slice culture. We further developed
these studies with in vivo experiments designed to compare and contrast
the safety and efcacy of replication competent recombinant wt-VSV and
replication restricted GTx-v401 in reducing tumor volume in a rat model
of glioma. In these studies, we found that the use of wt-VSV to treat intra-
cranial glioma resulted in an overwhelming encephalopathy and caused
unacceptable morbidity and mortality. In sharp contrast to these results,
administration of GS-VSV directly to the intracranial tumor bed was well
tolerated and effective at reducing the tumor load. In the highest plaque-
forming unit dose of GS-VSV tested (10
9
pfu, 5 logs higher than the lowest
dose of wt-VSV tested), there was little morbidity and no mortality associ-
ated with administration to the tumor site. A 71% reduction of tumor sur-
face area was noted in the GS-VSV treated group versus controls. We also
found no signicant differences between treated and control animals with
respect to weight loss, neurological changes, or neurohistopathological
changes at all doses tested. GS-VSV appears to be safe and effective when
used in vivo to treat intracranial glioma and warrants further development
as an adjuvant therapy.
414. COMINATION OF IMATINIB MESYLATE (STI-571,
GLEEVEC) AND TEMOZOLOMIDE (TEMODAR) DISPLAYS
INCREASED ACTIVITY AGAINST GLIOMA XENOGRAFTS
J. Rich,
1
S. Keir,
2
S. Sathornsumetee,
2
D. Reardon,
2
D. Bigner,
3
and H.
Friedman
2
; Departments of
1
Medicine,
2
Surgery, and
3
Pathology, Duke
Temozolomide is an orally available methylator now widely used in
glioma therapy. Although temozolomide has activity against malignant
gliomas, development of resistance is common. We have sought to increase
the activity of temozolomide in these cancers. Imatinib mesylate is a novel
low-molecular-weight ATP-mimetic inhibitor of several tyrosine kinases,
including platelet-derived growth factor receptors (PDGFRs). Although
imatinib mesylate has been relatively inactive in monotherapy trials against
gliomas, imatinib mesylate may offer a benet in increasing the efcacy of
temozolomide through increased tumor accumulation by decreasing inter-
stitial uid pressure and blocking retrograde blood-brain barrier transport-
ers as well as disrupting tumor survival mechanisms. The combination of
imatinib mesylate (200 mg/kg 5 days starting two days before temozolo-
mide dosing) and temozolomide (single dose at 0.1 LD
10
) was well tolerated
by athymic mice without signicant weight loss. Athymic nude mice bear-
ing established subcutaneous human glioma xenografts displayed additive
benet in time to reach 5 original tumor volume (24.8 days temozolo-
mide alone, 2.6 imatinib alone, 26.3 with the combination). The impact of
the combination was signicantly greater on the survival of mice bearing
intracranial xenografts (control 22 days, imatinib mesylate 25 (P 0.1),
temozolomide 49 (P 0.001), combination 60 (P 0.001 vs. control,
P 0.018 vs. temozolomide alone). Additionally, only mice treated with
the combination became long-term survivors (3/10). Current studies are
conrming PDGFR inhibition and exploring mechanism of the additional
benet. These results suggest that gliomas may exhibit greater sensitivity
to temozolomide when combined with imatinib mesylate. The combina-
tion of imatinib mesylate and temozolomide offers combinatorial benet
and is now in development as a clinical trial. This work was supported
by the Pediatric Brain Tumor Foundation of the United States, Accelerate
Brain Cancer Cure, Southeastern Brain Tumor Foundation, and NIH grant
NS047409 (J.N.R.). J.N.R. is a Damon Runyon-Lilly Clinical Investigator
and a Sidney Kimmel Cancer Foundation Scholar.
415. IMATINIB MESYLATE (STI571) INHIBITION
OF MALIGNANT GLIOMA CELLS IS INCREASED BY
MODULATION OF THE MAPK PATHWAY
H. Ren,
1
C. Walker,
3
L. Pazmany,
4
S. Pelech,
5
and N. Rainov
1,6
;
1
Department of Neurological Sciences, University of Liverpool,
Liverpool, UK; Department of Immunology, Harbin Medical University,
Harbin, PR, China;
3
JK Douglas Laboratories, Clatterbridge Cancer
Research Trust, Wirral, UK;
4
Academic Rheumatology Unit, University
of Liverpool, Liverpool, UK;
5
Kinexus Bioinformatics Corporation,
Vancouver, Canada;
6
Neurosurgery, The Walton Centre for Neurology
and Neurosurgery NHS Trust, Liverpool, UK
This study investigated mechanisms of action of imatinib mesylate
(STI571, Gleevec), a novel receptor tyrosine kinase signal transduction
inhibitor, in human malignant glioma cells. Real-time PCR (RT-PCR) was
carried out to quantitate the expression of PDGFa and PDGF receptors
(PDGFRa and PDGFR) and the stem cell factor receptor c-Kit, putative
targets of the drug, during imatinib treatment. Multi-immunoblot (Kinet-
works) differential protein kinase and phosphorylation-specic proling
was performed on imatinib-treated and control glioma cells. The malig-
nant human glioma cell lines U87MG and LNZ308 expressed PDGFa
and PDGF receptors, but not c-Kit. T98G human malignant glioma cells
expressed neither PDGF receptors nor c-Kit. RG human malignant glioma
cells expressed both PDGF receptors and c-Kit. Treatment with imatinib
caused dose-dependent downregulation of PDGFR in all expressing cell
lines, while PDGFRa expression was less affected. Expression of c-Kit in
RG cells was also downregulated by imatinib treatment in a dose-dependent
manner. Phosphorylation-specic multi-immunoblot (Kinetworks

KPSS) in
U87MG glioma cells treated with imatinib showed signicant functional
activation (up to 325% of control) of MAP kinases ERK1 and ERK2,
compared with untreated control cells. Phosphorylation activity of other
kinases in the MAPK signaling pathway, such as MEK1 and MEK2, and
of kinases outside this pathway, was downregulated by imatinib. Quantita-
tive RT-PCR in U87MG cells showed upregulation of the ERK phospha-
tase MKP-1 and downregulation of the ERK phosphatase MKP-3 after
treatment with imatinib. Finally, simultaneous specic inhibition of ERK
kinases during imatinib treatment using the MAPK inhibitor PD098059
signicantly increased the toxicity of imatinib in U87MG glioma cells. In
conclusion, our data show that inhibitory effects of imatinib on malignant
glioma cells are mediated at least in part by the MAPK signaling pathway.
Pharmacological inhibition of components of the MAPK signaling pathway,
as suggested by data from a phosphorylation-specic assay, can result in
increased toxicity of imatinib and in improved killing of glioma cells.
416. NOVEL POLYMERIC MICELLE DRUG CARRIER
SYSTEMS FOR BRAIN TUMOR THERAPY
Y. Ino,
1
Y. Guan,
1
N. Nishiyama,
2
H. Cabral,
3
K. Kataoka,
3
and T.
Todo
1
;
1
Department of Neurosurgery, Hospital;
2
Center for Disease
Biology and Investigative Medicine, Graduate School of Medicine, and
3
Department of Material Science and Engineering, Graduate School of
Engineering, The University of Tokyo, Tokyo, Japan
The use of polymeric micelles is one of the promising modalities of
macromolecular carrier systems. The particle size of polymeric micelles
(~50 nm) is smaller than that of other macromolecular carriers, and they
are therefore not easily trapped by the reticular systems during circulation.
The long circulation time will allow them to accumulate effectively in the
solid tumor through the enhanced permeability and retention (EPR) effect.
Doxorubicin or cisplatin carried by polymeric micelles has shown higher
antitumor activities than the drugs alone. Diaminocyclohexane platinum
(Dach-platin) is a second-generation platinum based anticancer drug which
is highly hydrophobic and is toxic when administered systemically. We have
developed a new polymeric micelle incorporating Dach-platin (Dach-Pt/
m) via the polymer-metal complex formation between Dach-platin and
poly(ethylene glycol)-poly(aspartic acid) block copolymers (PEG-P[Asp]).
The Dach-Pt/m was designed so that it would accumulate in the tumor
and be released only after reaching the tumor. The efcacy of Dach-Pt/m
was tested in Neuro2a (murine neuroblastoma) subcutaneous and intrace-
rebral tumor models. Oxaliplatin, a less toxic derivative of Dach-platin,
was also used as a control. All animal experiments have been approved
by the review committee of the Tokyo University. The maximum tolerated
dose (MTD) of Dach-Pt/m in A/J mice was 1.75mg/kg and similar to that
of oxaliplatin when administered from the tail vein three times every other
day. The toxicity was presumably due to a spontaneous decay of the micelle
causing accumulation of Dach-Pt in the liver followed by liver dysfunction.
Nonetheless, Dach-Pt/m was more effective in inhibiting the Neuro2a sub-
cutaneous tumor growth than oxaliplatin at the MTD level. Dach-Pt/m was
also effective in the Neuro2a intracerebral tumor model and prolonged the
survival of tumor-bearing mice compared with oxaliplatin. These results
suggest that the polymeric micelle macromolecular carrier system may be
useful for the treatment of brain tumors. Optimization of dosing schedules
may further enhance the efcacy of Dach-Pt/m.
417. SPECIFIC TRANSLOCATIONS OF CHROMOSOMES 11
AND 22 IN RECURRENT MALIGNANT GLIOMAS
A.C. Scheck,
1
L. Panyon,
1
N.C. Hank,
1
and G. Martinez
2
;
1
Neuro-
Oncology Research, Barrow Neurological Institute;
2
Biological Sciences,
Paradise Valley Community College, Arizona State University West,
Phoenix, USA
Malignant gliomas are typically treated with surgery, radiation, and
chemotherapy. Despite this, these tumors recur and are resistant to addi-
tional treatment. We have previously demonstrated that cells selected for
resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in vitro or in
vivo (recurrent tumor from patients treated with BCNU) are near-diploid
with over-representation of chromosome 7 and regions of chromosome 22.
To identify the over-represented regions of chromosome 22, we analyzed
cells from several primary/recurrent tumor pairs prior to, and following
selection for resistance to 10 g/ml of BCNU. Fluorescent in situ hybrid-
ization (FISH) using bacterial articial chromosome (BAC) probes allowed
us to map specic chromosomal aberrations in these cells. FISH analyses
allowed us to map the over-represented region to 22q12.3-13.31. In addi-
tion, we have identied 3 specic translocations in cells from recurrent
tumor involving chromosomes 22 and 11. We mapped the chromosome
11 breakpoints to within 1.5 Mbp and the chromosome 22 breakpoints
to less than 82 kbp. The rst translocation occurs between the telomeric
side of 22q12.3 and centromeric edge of 11q23.1. The second translocation
involves sequences on the 22q12.1/22q12.2 and 11q23.1/11q23.2 borders.
The third translocation occurs between the telomeric sides of 22q11.1 and
11q23.3. Additional work has shown that these translocations are fre-
quently reciprocal and found in addition to normal copies of the chromo-
somes. We have also found them in parafn-embedded tissue from recur-
rent tumor but not in tissue from the same patients primary tumor. Further,
in vitro selection for cells resistant to BCNU also selects for cells with these
translocations; however, in vitro treatment of cells from primary tumor
cannot induce these translocations. Preliminary work suggests that radia-
tion treatment may be a causative event in the formation of these transloca-
tions. This work suggests that one or more of these translocations provides
the cell with a selective advantage that contributes to therapy resistance or
to the growth of therapy resistant cells.
418. ENCOURAGING RESULTS FOR A NOVEL
CHEMOTHERAPEUTIC REGIMEN IN NEWLY DIAGNOSED
E. Arenson, J. Bank, M. Pierick, C. Greenwald, T. Fullagar, and J.
McVicker; Colorado Neurological Institute and Swedish Medical Center,
Englewood, Colorado, USA
Substantial benefit from chemotherapy of cancer requires effective
combinations; nevertheless, monotherapy with temozolomide (TMZ) has
emerged as the standard treatment of glioblastoma multiforme (GBM).
We report results of treatment of GBM with the novel combination of
BCNU, irinotecan (CPT11) and TMZ (BITE). Four patients were excluded
from this treatment because of expected survival 3 months, absence of
caregiver, or 24-h care requirement. Thirty-seven patients with newly diag-
nosed GBM were treated between August 1999 and October 2002. Mean
age was 53.4 years. Thirteen patients had bilateral and/or multifocal dis-
ease. Nine had gross total resection, 27 subtotal resection, and 1 biopsy, as
determined by early post-operative contrast MRI. Treatment consisted of
three courses of CPT11 (400 mg/m
2
1) and TMZ (200 mg/m
2
5) given
every 21 days during standard radiotherapy (RT) (phase I) to which BCNU
(40 mg/m
2
3) was added after RT for up to 6 monthly courses as tolerated
(phase 2). Three patients did not complete phase 1 because of patient choice,
neurological decline, or death from intratumoral hemorrhage unrelated to
treatment. Two additional patients did not proceed to phase 2 because of
poor performance status. Thirty-two patients received 115 courses of BITE
(mean 3.4 courses) and had episodes of grade 3/4 toxicities as follows: GI
12%, neutropenia 42%, and thrombocytopenia 11%. There were 2 deaths
during phase 2 unrelated to disease progression: one disseminated CMV
and one bacterial pneumonia. One patient survived an atypical mycobacte-
rial pneumonitis, and one survived an episode of BCNU pneumonitis. With
minimum follow-up of 30 months, mean survival is 19 months. Overall sur-
vival is 59% 1 year, 30% 2 years, and 19% 3 year3. Relapse-free survival
is 46% 1 year, 22% 2 years, and 11% 3 years. Six patients are alive (16%),
ve without evidence of disease and one with stable disease from 37 to 48
months post-diagnosis. For patients with initial gross total resection (24%),
relapse-free survival was 89% at 1 year, 89% 2 years, and 56% 3 years. We
conclude that (1) BITE is toxic, but effective, (2) polychemotherapy should
not be abandoned in GBM, (3) BITE deserves further study with efforts
to reduce toxicity, and (4) patients with gross total resection may survive
long-term with aggressive post-surgical treatment.
419. SALVAGE CHEMOTHERAPY WITH
CYCLOPHOSPHAMIDE FOR RECURRENT
TEMOZOLOMIDE-REFRACTORY ANAPLASTIC
ASTROCYTOMA
M. Chamberlain, D. Tsao-Wei, and S. Groshen; Preventive Medicine,
University of Southern California, Los Angeles, California, USA
We conducted a prospective phase 2 study of cyclophosphamide (CYC)
in adult patients with recurrent temozolomide-refractory anaplastic astro-
cytoma (AA) with a primary objective of evaluating 6-month progression-
free survival (PFS). Forty patients (28 men; 12 women) ages 26 to 57 years
(median 43), with recurrent AA were treated. All patients had previously
been treated with surgery and involved-eld radiotherapy. Additionally, all
patients were treated adjuvantly with temozolomide (TMZ) chemotherapy.
All patients were treated at rst recurrence with CYC administered intrave-
nously on 2 consecutive days (750 mg/m
2
/day) every 4 weeks (operationally
dened as a single cycle). Neurological and neuroradiographic evaluation
were performed every 8 weeks. All patients could be evaluated. A total of
215 cycles of CYC (median 2 cycles; range 212) was administered. CYC-
related toxicity included alopecia (all patients, 100%), anemia (5, 12.5%),
thrombocytopenia (6, 15%), and neutropenia (8, 20%). Four (10%)
patients required transfusion. Nine patients (22.5%; 95% CI, 11%39%)
demonstrated a neuroradiographic partial response, 16 patients (40.0%;
95% CI, 25%57%) demonstrated stable disease and 15 patients (37.5%;
95% CI, 23%54%) had progressive disease following two cycles of CYC.
Time to tumor progression ranged from 2 to 19 months (median, 4 months;
95% CI, 26 months). Survival ranged from 2 to 26 months (median, 8
months; 95% CI, 610) months). Six-month and 12-month PFS was 30%
and 8%, respectively. CYC demonstrated modest efcacy with acceptable
toxicity in this cohort of adult patients with recurrent anaplastic astrocy-
toma, all of whom had failed prior TMZ chemotherapy.
420. HIGH-DOSE BCNU WITH AUTOLOGOUS BLOOD
STEM CELL RESCUE: DEFINITIVE RESULTS OF A
PHASE 3 MULTICENTRIC STUDY IN SUPRATENTORIAL
COMPLETELY RESECTED GLIOBLASTOMA PATIENTS
TREATED WITH POST- OPERATIVE RADIOTHERAPY
M. Ben Hassel,
1
C. Linassier,
2
D. Figarella-Branger,
3
J.O. Bay,
4
H.
Bourgeois,
5
T. Lesimple,
1
J.P. Cottier,
6
L. Taillandier,
8
R. Delepine,
2

and D. Frappaz
7
;
1
Centre Eugne Marquis, Rennes;
2
CHRU Bretonneau,
Tours;
3
CHU La Timone, Marseille;
4
Centre Jean Perrin, Clermont-
Ferrand;
5
CHU La Mileterie, Poitiers;
6
Centre Alexis Vautrin, Nancy;
7
Groupe Hospitalier Piti-Salptrire, Paris;
8
Centre Lon Brard,
Lyon; Neuro-Oncology Group of the National French Federation of
Comprhensive Cancer Centers, FNCLCC ; France
A previously published phase 2 study with high-dose BCNU and autolo-
gous bone marrow graft added to post-operative radiotherapy suggested a
favourable trend in extensively resected adult glioblastoma patients. In the
present study patients were randomized after extensive surgery between
two groups: arm A, BCNU at 800 mg/m
2
followed by stem cell rescue on
day 3 and radiotherapy 4 weeks later (60 Gy and classical fractionation),
and arm B, radiotherapy (60 Gy, classical fractionation) followed by 80 mg/
m
2
BCNU at 6-week intervals during one year. Criteria of inclusion were as
follows: age 1665, supratentorial tumor site, glioblastoma histology, gross
total tumor resection, OMS performance status (PS) 0-2, adequate liver,
kidney, and lung functions, no pregnancy, no other malignant tumor, and
written consent. Sixty-nine patients only (median age 50.8 years) instead
of the 140 expected were included in the study between September 1997
and November 2001: 36 in arm A and 33 in arm B. Both post-operative
imaging and histology were subjected to review. Follow-up lasted up to
a data analysis made in December 2004 (3 years after the last inclusion).
Toxicity was recorded according to WHO classication. The study was
closed because of a prohibitive toxicity death rate above 10%: 1 case of
septic shock and 3 cases of severe lung brosis were recorded in arm A. One
reversible grade 4 liver toxicity was recorded in arm A. Denitive results
will be presented in terms of early and late toxicity, time to progression,
and survival. We conclude that favorable prognosis factors like age, exten-
sive resection, and PS, have a stronger inuence than a high-dose BCNU
chemotherapy regimen.
421. CARBON ION RADIOTHERAPY FOR MALIGNANT
GLIOMAS
J. Mizoe,
1
H. Tsujii,
1
A. Hasegawa,
1
R. Takagi,
1
T. Yanagi,
1
and K.
Takakura
2
;
1
National Institute of Radiological Sciences, Research Center
for Charged Particle Therapy, Chiba;
2
Neurosurgery, Tokyo Womens
Medical University, Tokyo; Japan
We conducted a study to investigate the effects of combined X-ray,
ACNU, and carbon ion radiotherapy for malignant gliomas. Between Octo-
ber 1994 and February 2002, 48 patients with histologically conrmed
malignant gliomas (16 anaplastic astrocytoma and 32 glioblastoma multi-
forme) were enrolled into the phase 1/2 clinical study of combined X-ray,
ACNU, and carbon ion radiotherapy. Their age range was from 18 to 78,
and the mean age was 53 years. By gender, they comprised 29 males and
19 females. The loci included 22 frontal lobe, 10 temporal lobe, and so
on. Twenty-seven patients underwent partial resection, 8 subtotal resec-
tion, and 8 macroscopic total resections prior to radiotherapy. Treatment
involved the application of 50 Gy/25 fractions/5 weeks of X-ray, followed
by carbon ion radiotherapy at 8 fractions/2 weeks. ACNU of 100 mg/m
2

were administrated concurrently in the rst and fourth or fth weeks of
X-ray therapy. Carbon ion dose was escalated from 16.8 to 24.8 GyE on
10% incremental steps after conrmation of the safety of each dose given
previously. There were 9 cases with grade 2 acute reaction in the skin but
there was no grade 2 or higher reaction in the brain. The late reactions
included 2 cases of grade 2 brain morbidity (RTOG/EORTC) and 3 cases
of grade 2 brain reaction (LENT/SOMA, MRI) out of 48 cases. There were
no grade 3 or higher-grade reactions until the date of analysis. Median
survival time (MST) of AA was 35 months and that of GBM 17 months.
In the AA patients, MST was 20 months for the low-dose group (16.8 ~
20.0 GyE, 6 patients) and 40 months for the high-dose group (22.4 ~ 24.8
GyE, 10 patients) (P 0.0382). MST of GBM patients was 7 months for
the low-dose group (16.8 GyE, 7 patients), 19 months for the middle dose
group (18.4 ~ 22.4 GyE, 23 patients), and 24 months for the high-dose
group (24.8 GyE, 5 patients) (P 0.031). In recursive partitioning analy-
sis (RPA), two years overall survival rate of class I (5 patients) was 80%,
class II (5 pts) 60%, class III (12 pts) 58%, class IV (14 pts) 21%, class
V (6 pts) 50%, and class VI(6 pts) 33%. Results of combined therapy of
X-ray, ACNU, and carbon ion radiotherapy showed the potential efcacy
of carbon ion radiotherapy for malignant gliomas in terms of the improved
survival rate in those patients who received higher carbon doses. Based
on these results, a new protocol for malignant gliomas using carbon ion
radiotherapy alone was initiated.
422. EFFICACY OF RADIATION THERAPY ON SEIZURES IN
LOW- GRADE ASTROCYTOMAS
R. Sofetti, M. Borgognone, A. Ducati, P. Gaviani, E. Laguzzi, M.
Nobile, U. Ricardi, and R. Rud; Neuroscience, University of Torino,
Torino, Italy
The role of radiation therapy in low-grade astrocytomas is still contro-
versial, and seizure control represents the most relevant clinical problem.
There are some reports in the literature suggesting that radiation therapy
might be effective in reducing seizure frequency in patients with inoper-
able low-grade gliomas. The objective of this study was to analyze, in a
retrospective series of patients with supratentorial histologically veried
grade II astrocytomas and persisting seizures despite conventional AEDs,
the response of epilepsy to conventional radiotherapy. The study popula-
tion (19852001) included 25 patients, ages 20 to 54 years (median 35), 16
men and 9 women. Previous surgery consisted of a biopsy in 9 patients, a
partial/subtotal resection in 13 patients, and a total resection in 3 patients.
The seizure frequency varied from 1 per day to 1 per month, and the
type was as follows: partial simple in 9, partial complex in 9, and general-
ized in 7. Twenty-two tumors were non-enhancing on CT or MRI, whereas
3 only were enhancing. Patients received external radiotherapy either adju-
vantly or at tumor progression in conventional fractionation, with total
doses ranging from 40 to 60 Gy (median 54). Overall, 19 patients (76%)
had a signicant reduction (50% decrease) in seizure frequency, with 2
patients seizure free, whereas 5 patients (20%) had no signicant change,
and 1 (4%) had an increase in seizures. Basing on Macdonalds criteria of
response, we observed 6 PR (24%), 17 SD (68%), and 2 PD (8%). When
correlating the clinical with the radiological response, among patients who
had a signicant reduction of seizure frequency (19), 4 (21%) had a PR, 13
(68%), an SD, and 2 (11%) a PD, whereas among patients who had no sig-
nicant change of seizure frequency (5), 2 (40%) had a PR, and 3 (60%) a
SD. In conclusion, this study conrms in a larger series of patients that con-
ventional radiotherapy is able to signicantly reduce the seizure frequency
in a high proportion of patients with grade II astrocytomas. Moreover, a
correlation does not seem to exist between the seizure reduction and tumor
response on CT or MRI.
423. OBSERVATIONS ON THE RELATIONSHIP BETWEEN
RADIOTHERAPY AND SURVIVAL IN PATIENTS WITH
LOW- GRADE GLIOMAS FROM THE SURVEILLANCE,
EPIDEMIOLOGY, AND END RESULTS (SEER) PROGRAM
DATA
B. Lally,
1
J. Baehring,
2
D. Zelterman,
3
and J. Knisely
1
;
1
Therapeutic
Radiology;
2
Neurosurgery & Neurology;
3
School of Public Health, Yale
University School of Medicine, New Haven, Connecticut, USA
Despite trials providing class I data, radiotherapys use in the manage-
ment of low-grade gliomas (LGG) remains controversial. The utilization of
radiotherapy and its relationship to survival was examined in the Surveil-
lance, Epidemiology, and End Results (SEER) program data. SEER is a
national cancer surveillance program that covers ~26% of the U.S. popula-
tion. SEER database patients with supratentorial, pathologically conrmed
LGG diagnosed between 1983 and 2001 who were 21 to 60 years of age
were evaluated. The relationship between early postoperative radiotherapy
(yes or no) and various other prognostic variables was evaluated. These
included age (40 vs. 40), extent of surgery (biopsy vs. subtotal vs. total
resection vs. surgery, NOS), and histology (oligodendroglioma, oligoastro-
cytoma, and astrocytomawhich included brillary, protoplasmic astro-
cytomas and astrocytoma, NOS). A total of 4210 patients were in the nal
pool for analysis. SAS version 8.1 (SAS Institute, Cary, N.C.) was used for
all analyses. The
2
test was used to test for interaction between radiother-
apy and other variables. Kaplan-Meier methods were used to estimate crude
survival from time of diagnosis until death from any cause. Multivariable
Cox proportional hazards models were used to calculate adjusted hazard
ratios (HR) to assess the importance of radiotherapy as an independent
predictor of survival. The HR for age 40 was 2.124; radiotherapy was
more frequently used in patients 40 (P 0.0001). The HR for oligo-
dendroglioma was 0.658 (P 0.0001), and the HR for astrocytoma was
1.613 relative to oligoastrocytoma; radiotherapy was more frequently used
for astrocytomas (P 0.0001). Survivals were increased for complete and
subtotal resections compared to biopsy and surgery, NOS. Radiotherapys
use was associated with an HR of 1.509; it was more commonly used with
less complete surgical resection. Median survivals for astrocytoma, oligoas-
trocytoma, and oligodendroglioma histologies with or without the use of
postoperative radiotherapy were (in years) 3.2 versus 7.5; 6.4 versus not
yet reached; and 10.0 versus 14.6, respectively. It is difcult to discern any
benet from early radiotherapy as a result of this retrospective analysis.
424. ENHANCEMENT OF RADIOSENSITIVITY OF HUMAN
MEDULLOBLASTOMA CELLS BY HISTONE DEACETYLASE
INHIBITOR VALPROIC ACID IS ASSOCIATED WITH THE
ACTIVATION OF P21 AND DOWN-REGULATION OF C-MYC
AND SURVIVIN GENES
X. Li,
1
Q. Shu,
1
A. Munshi,
2
Y. Xia,
3
E. Chang,
1,2
S. Woo,
2
and
C. Lau
1
;
1
Pediatrics and Texas Childrens Cancer Center, Baylor
College of Medicine, Houston, Texas, USA;
2
Experimental Radiation
Oncology and Division of Radiation Oncology, The University of Texas
M.D. Anderson Cancer Center, Houston, Texas, USA;
3
Cancer Center,
Radiation Oncology, Sun Yat-sen University, Guangzhou, China
Histone deacetylase (HDAC) inhibitors are emerging therapeutic
agents capable of disrupting critical cellular processes in cancer cells. One
particular novel HDAC inhibitor that has great potential of being quickly
translated into clinical trials is valproic acid (VPA), which is an established
anti-convulsant drug with well-known safety proles. VPA can also pass
through the blood-brain barrier, making it a more attractive agent for treat-
ment of malignant brain tumors such as medulloblastoma (MB). Our previ-
ous study has demonstrated that VPA at clinically achievable doses of 0.6
and 1 mM, suppressed cell proliferation, arrested cells in G
0
/G
1
phases,
induced apoptosis, reduced tumorigenicity in SCID mice, and is also active
in MB subcutaneous xenograft models. To investigate the ability of VPA in
modulating cellular responses to ionizing radiation in MB cells, four MB
cell lines (D283-MED, DAOY, MHH-MED-1, and MEB-MED-8A) were
pretreated with VPA (0.1, 0.2, 0.6, and 1 mM) for 7 days, followed by
irradiation at doses of 2, 4, and 6 Gy. Our results showed that VPA (0.2,
0.6, and 1 mM) not only suppressed the colony-forming efciency by itself,
but also enhanced the radiosensitivity in all four MB cell lines (P 0.01).
Complete suppression of colony formation was achieved by 4 Gy irradia-
tion in D283-MED and MEB-MED-8A cells pretreated with 1 mM VPA,
an effect that was not seen with 6 Gy irradiation alone. The mechanisms
underlying VPA-induced radiosensitization were further investigated by
examining the expressions of genes involved in cell cycle regulation and
apoptosis. Upon treatment with VPA, the expression of p21 was increased,
while that of c-Myc and the anti-apoptosis gene survivin were signicantly
reduced. Taken together, these results suggest that activation of cell cycle
inhibitor and inhibition of DNA repair and anti-apoptosis genes are the
mechanisms underlying radiation-enhancing effects of VPA and support the
use of VPA as a radiosensitizer in order to reduce the radiation doses and
thereby the toxicities on developing brains.
425. LOCAL RADIOTHERAPY AFTER RESECTION OF
SINGLE BRAIN METASTASIS
S. Kinhult,
1
A. Gustavsson,
1
and E. Kjelln
1
;
1
Department of Oncology,
Lund University Hospital, Lund, Sweden
Patients with a single, resectable brain metastasis can have a relatively
good prognosis, with median survival of approximately 1 year. Radio-
therapy to the brain can have serious side effects with high risk of leuken-
cephalopathy and dementia after 0.5 to 1 year. So far, the value of post-
operative whole-brain radiotherapy has not been established. Therefore,
to minimize these late side effects of radiotherapy, patients operated for
single brain metastasis were given postoperative local radiotherapy instead
of whole-brain radiotherapy. From 2000, patients who were referred for
postoperative radiotherapy after resection of single brain metastasis were
given a planned dose of 25 Gy in 5 fractions to the metastasis site with a
margin of 2 cm. Twenty-four patients were treated according to the pro-
tocol, 11 men and 13 women. The primary tumors were 8 lung cancer, 4
breast cancer, 4 unknown primary, 2 renal cancer, 2 malignant melanoma,
and 4 gastrointestinal tumors. Median age at treatment was 58.5 (range,
4879) years. Median time from primary diagnosis to brain metastasis was
22.2 (0160) months. Median time from diagnosis of brain metastasis to
surgery was 32 (683) days and from surgery to radiotherapy 36.5 (2080)
days. The planned dose was delivered to 22 patients, one patient had 20
Gy in 4 fractions, and one had 20 Gy in 5 fractions. The radiotherapy
was given with mask xation and in 7 patients after 3D dose planning.
Intracranial recurrence was seen in 14 patients; only 3 of these patients had
recurrence only outside the irradiated volume. Six patients did not have any
intracranial recurrence, and 3 were lost to follow-up, but 1 of these 3 was
alive after 3 years. Median time from radiotherapy to intracranial progres-
sion was 6.7 (1.525.3) months. Three patients had a second operation for
the same or a second brain metastasis. Eight patients were given cranial
radiotherapy a second time, 30 Gy in 10 to 15 fractions to the whole brain.
Median survival from surgery was 10.8 (1.647.4) months, and from start
of radiotherapy 9.8 (0.245.9) months. Seven patients were alive at time
of analysis. Five of them had no intracranial recurrence, with a median
survival from surgery of 11.0 (1.647.4) months. A majority of the patients
with a resected brain metastasis recur within the irradiated volume after
postoperative radiotherapy. This does not support giving whole-brain radi-
otherapy postoperatively after resection of single brain metastasis. Reduc-
ing the irradiated volume diminishes the risk of late side effects, especially
cognitive disturbances.
426. STEREOTACTIC RADIOSURGERY FOR ATYPICAL/
ANAPLASTIC MENINGIOMAS
J.A. Takahashi, H. Kano, N. Katsuki, M. Shirahata, M. Ohno,
N. Hashimoto, N. Araki, and M. Hiraoka; Neurosurgery, Kyoto
University Hospital, Kyoto, Japan
Atypical and anaplastic meningiomas frequently recur in the relatively
short-term after surgery, even if they are radically resected. We have fol-
lowed such postoperative cases by short-interval repeated MRI and have
performed stereotactic radiosurgery (SRS) toward progressive tumors as
salvage therapy. We report these results of SRS in high-grade meningioma
in comparison with low-grade meningiomas. We reviewed 15 meningioma
patients with 28 lesions treated by SRS at Kyoto University Hospital between
1997 and 2001, all of whom underwent initial surgery and received SRS for
tumor progression as postoperative salvage therapy. They included 5 low-
grade meningiomas, 9 atypical ones, and one anaplastic type. The mean
tumor volume was 6.35 cm
3
(03.5118 cm
3
), and the mean marginal dose
was 18.1 Gy (1220 Gy). After a mean follow-up period of 30.9 months
(2472 months), 8 cases had tumor progression within the SRS eld, and
6 had out of the SRS eld. Cases with tumor progression had atypical/
anaplastic meningiomas except one case with low-grade meningioma in the
skull base. In atypical/anaplastic meningiomas, the mean time to progres-
sion after SRS was 12.3 months, and the mean marginal dose was 17.3
Gy. Five of 9 lesions, which were treated by less than 20 Gy SRS, had local
recurrence within the SRS eld. The marginal dose less than 20 Gy was a
statistically signicant predictor for a short-term progression in high-grade
meningiomas (P 0.0029). The two-year progression-free survival ratio
in cases 20 Gy and 20 Gy was 40% and 57%, respectively. Extra-eld
tumor progression was due to CSF dissemination, meningiomatosis, and
diffuse dural invasion. While tumor progression of low-grade meningiomas
was controlled by relatively low-dose SRS (1518 Gy), cases of high-grade
meningiomas were recommended to receive SRS with marginal dose of
more than 20 Gy.
427. BIODISTRIBUTION AND INTERNAL DOSIMETRY
OF THE 188-RE LABELED HUMANIZED MONOCLONAL
ANTIBODY H-R3 ADMINISTERED LOCO-REGIONALLY TO
PATIENTS WITH HIGH-GRADE MALIGNANT GLIOMAS:
PRELIMINARY RESULTS
L.A. Torres Aroche,
1
M. Coca,
1
A. Casaco,
2
G. Lopez,
3
I. Garcia,
3

A. Perera,
1
J. Batista,
1
A. Hernandez,
1
and Y. Pea
1
;
1
Nuclear Medicine,
Center for Clinical Research,
2
Clinical Research, Center of Molecular
Immunology, and
3
Neurosurgery, International Center of Neurological
Restoration, Havana, Cuba
Favorable clinical response after direct infusion of 90-Y or 131-I labeled
anti-tenascin Mabs into the postoperative tumoral bed of patients with
malignant gliomas has been found. A clinical trial was performed to evalu-
ate the biodistribution, internal radiation dosimetry, toxicity, the maximal
tolerated dose (MTD), and any clinical effect after the loco-regional radio-
immunotherapy (RIT), using the humanized MAb h-R3, labeled with188-
Re. A phase 1 dose escalation trial was performed by administrating into
the post-operative cavity through an indwelling catheter a single dose of the
h-R3 MAb directed against epidermal growth factor receptors (EGFR).
The study was reviewed and approved by the ethics committees of all the
involved institutions. Five patients have been included; they had partial
tumor resections and overexpressed the EGFR. Patients were treated with
3 mg of MAb labeled with 10 or 15 mCi of 188-Re after signing the written
informed consent. SPECT and planar images as well as multiple blood and
urine samples were collected up to 24 h after injection. Biodistribution was
computed from scintigraphic images, and the absorbed doses were esti-
mated using the MIRD methodology at organ and voxel level. Data process-
ing and statistical analyses were performed using the SPSS and Microcal
Origin v6.0 software packages. The effective half-life of the 188-Re H-R3
in the tumoral bed ranged from 7.3 to 14.4 h (mean, 8.4 2.8 h). The liver,
kidneys, and urinary bladder showed the highest uptake of the compound
leaving the tumoral bed. The mean absorbed dose in the tumor ranged from
13.9 Gy to 68.4 Gy, and the maximum doses ranged from 26.9 Gy to 136.2
Gy. The maximum absorbed dose for liver, kidneys, and urinary bladder
was less than 2 Gy in all patients. Transitory acute side effects following
treatment were headache, seizures, and worsening of pre-existing neurolog-
ical symptoms. Two patients developed stable disease during 3 months, and
2 GBM patients are practically asymptomatic and in complete remission
after one year of treatment. The other GBM patient cannot yet be evaluated
after one month of treatment. The preliminary results of this study strongly
suggest that loco-regional radioimmunotherapy of high-grade glioma using
the h-R3 MAb labeled with 188-Re may be safe and constitute a promising
therapeutic approach for these patients.
428. BRAINSTEM GLIOMAS IN ADULT: PROGNOSTIC
FACTORS AND RESULTS IN A SERIES OF WESTERN
ITALIAN COOPERATIVE GROUP
L. Fariselli, A. Salmaggi, I. Milanesi, E. Lamperti, A. Boiardi,
C. Maccagnano, E. Laguzzi, R. Ruda, and R. Sofetti; Radiotherapy,
Istituto Nazionale Neurologico C. Besta, Milan, Italy
The charts of 35 patients with brainstem gliomas were reviewed to
dene prognostic factors and to evaluate the effect of combined radio-
chemotherapy treatment. Mean age at onset was 39 years (1655). The
main presenting symptoms were diplopia (40%), paresis (30%), and ataxia
(10%). At MRI, 18 patients (51%) had diffuse inltrative tumor, in 15
patients (43%) diffuse or local contrast-enhancement, and in 2 patients
(6%) focal cystic-necrotic areas were described. MR-spectroscopy was
positive in all analyzed patients, and quantitative PET was performed in 9
patients. Twenty-four patients had surgical intervention; conformal radio-
therapy was performed in all patients (total dose range, 4854 Gy, 1.8 Gy/
fraction) associated to chemotherapy (temodal 250 mg/day weekly, max 5
cycles) in 20 patients. Overall median survival was 40 months. On multi-
variate analysis, the duration of symptoms before diagnosis, the absence of
contrast-enhancement, and histological diagnosis were conrmed to have
prognostic signicance on survival. Neurological conditions and/or radio-
logical response were improved in 50% of the cases, while they were stable
in 35% after treatment. Adult brain stem gliomas resemble supratentorial
gliomas: Radiotherapy associated to chemotherapy seems to have potential
chances of temporary improvement of neurological functions.
429. RADIOTHERAPY FOR PRIMARY BRAIN TUMORS:
CLINICAL POTENTIAL OF NOVEL TREATMENT
TECHNIQUES
C. Nil, H. Struikmans, and J. van Santvoort; Radiation Oncology,
Medisch Centrum Haaglanden, The Hague, The Netherlands
Radiation dose to the normal brain restricts the total radiation treat-
ment dose given for a primary brain tumor. In the last decade radiation
techniques have evolved to an approach which reduces the normal tissue
irradiation by optimizing the shape and beam direction of a given radia-
tion beam. These techniques are known as 3-dimensional conformal radio-
therapy (3dCRT) and are the current standard in radiation treatment for
primary brain tumors. The latest technical development in radiotherapy is,
apart from optimal shaping of the beam contour, the computerized modula-
tion of the dose content of a radiation beam, so-called intensity modu-
lated radiation therapy, or IMRT. The aim of these techniques is to increase
the effect of sparing normal tissue that has been achieved by 3dCRT. We
evaluated the possible clinical advantage of IMRT by comparing the actu-
ally given 3dCRT plan with an IMRT plan for 8 patients with low-grade
glioma, mean tumor volume 174 cm
3
(72329). We compared the volumes
of normal brain tissue irradiated to a total dose of respectively 20%, 40%,
and 60% of the prescribed treatment dose. A substantial reduction of brain
tissue irradiated to these dose levels (for 3dCRT 69 [4287], 40 [1854],
and 21 [430] cm
3
, respectively) was achieved by IMRT (42 [2755], 15
[824], and 5 [210] cm
3
), even for larger tumor diameters. The impact
of IMRT on the dose distribution for the primary tumor was small (mean
volume receiving at least 95% of the therapeutic dose: 93% [9098] vs.
100% for 3dCRT) and, in light of the therapeutic gain by reducing the
radiation dose to the normal tissue, not of any clinical relevance. Achiev-
able radiation doses to the tumor, given by IMRT without an increase of
risk in neurotoxicity are calculated from our data and the known data from
the literature. The clinical use of IMRT in the treatment for primary brain
tumors may lead to a substantial dose escalation without increasing the
potential radiation toxicity to the normal brain. Phase 2 studies, carefully
undertaken, should be performed to investigate the therapeutic potential of
IMRT in the treatment for primary brain tumors.
430. THE POSITIVE EFFECT OF RADIOTHERAPY DOSE
ESCALATION AND CHEMOTHERAPY ON THE SURVIVAL
OF GLIOBLASTOMA PATIENTS
L. Mangel,
1
L. Sipos,
2
J. Julow,
3
J. Havel,
3
T. Major,
1
G. Nmeth,
1
and
J. Fodor
1
;
1
Department of Radiotherapy, National Institute of Oncology,
Budapest;
2
National Institute of Neurosurgery, Budapest;
3
Department
of Neurosurgery, St. Johns Hospital, Budapest; Hungary
The aim of our retrospective study was to investigate novel therapeu-
tic approaches on the survival of glioblastoma patients. We analyzed the
data of 102 glioblastoma patients who were treated with denitive radio-
therapy (RT) between 1997 and 2003; the short palliative RT courses were
excluded. We examined the effect of age (70 vs. KPS 70), type of surgery
(radical vs. partial), T-stage (T1 vs. T24), adjuvant chemotherapy (CHT)
with Temodal and/or BCNU (yes vs. no), and RT dose escalation (biologi-
cal equivalent dose 60 Gy vs. 60 Gy) on survival. The RT dose escala-
tion consisted of hypofractionated regimen of 60/2.5 Gy, or interstitial AL
boost, or conventional RT of 66 Gy total dose. The treatment selection
was based upon patient and tumor characteristics, individually. Uni- and
multivariate analyses were performed. Good performance status and T1
stage had positive effect on survival. The median survival times (MST) were
14 versus 9 months (P 0.0004) and 14 versus 10 months (P 0.0024),
respectively. The type of surgery and the age showed a non-signicant ten-
dency, the MSTs were 13 versus 10 months (P 0.0724) and 12 versus
10 months (P 0.0724), respectively. The addition of CHT and RT dose
escalation signicantly prolonged the survival, the MSTs were 14.5 versus 8
months (p0.0001) and 13 versus 8.5 months (P 0.0001). Seven patients
were alive 2 years after initial diagnosis, 6 of them had been treated with
CHT, and all of the 2-year survivors received higher RT doses. Multivariate
analysis revealed that good KPS (P 0.0280; RR, 0.59; CI, 0.360.94),
low T-stage (P 0.0383; RR, 0.63; CI, 0.40.98), RT dose escalation (P
0.0009; RR, 0.46; CI, 0.290.73), and the addition of CHT (P 0.0017;
RR, 0.48; CI, 0.30.76) had independent positive effect on survival. This
study indicates that in addition to CHT the RT dose escalation may have
therapeutic benet. However, further investigation is required to dene the
optimal forms of RT dose escalation in glioblastoma patients with different
prognostic factors.
431. MAINTENANCE OF NEUROPSYCHOLOGICAL
FUNCTION IN CHILDREN AND YOUNG ADULTS WITH
BENIGN AND LOW- GRADE BRAIN TUMORS TREATED
PROSPECTIVELY WITH HIGH-PRECISION FOCAL
CONFORMAL RADIOTHERAPY
R. Jalali,
1
S. Goswami,
2
N. More,
1
R. Sarin,
1
M. Siddha,
3
R. Kamble,
1

and S. Parab
3
;
1
Radiation Oncology;
2
Clinical Psychology;
3
Brain Tumor
Foundation of India, Mumbai, India
The aim of our study was to present prospective neuropsychological
data at baseline and follow-up in children and young adults with benign and
low-grade gliomas treated with focal stereotactic conformal radiotherapy
(SCRT). Twenty-six patients (age 425 years) with residual/progressive
benign and low-grade tumors considered suitable for SCRT underwent a
detailed neuropsychological and cognitive testing at baseline before starting
RT and subsequently at 6 months and 24 months (and every year thereafter)
after SCRT. Intelligence quotient (IQ) was measured by an age-adjusted and
validated WISC giving verbal quotient (VQ), performance quotient (PQ),
and global quotient (GQ). For patients more than 17 years, memory quo-
tient (MQ) was measured by Wechsler memory scale (WMS). VITHOBA
battery was employed for blind children to assess PQ. Anxiety was measured
by C1 and C2 scales and Hamilton anxiety rating scale (HARS; for adults),
and depression was measured by Hamilton depression rating scale (HDRS;
for adults). Cognition was measured by LOTCA battery (max value, 119)
and quality of life by Health Utility Index (HUI; normal score: very good,
7; poor, 31). Mean baseline global-IQ (normal 90109) of patients before
starting RT was 80 (range, 33129), which improved to 87 and 92 respec-
tively at 6 and 24 months following SCRT. The corresponding mean values
for VQ and PQ at similar timescale were 82, 87, and 89 and 82, 93, and 106
respectively. Memory remained maintained at 6 months and 2 years after
SCRT, with mean values of 97 and 103 (range, 100106) compared to mean
baseline value of 83 before RT. In 3 blind children, PQ recorded by Vithoba
battery revealed a reduction of mean value 94 at 6 months and 77 at 2
years after RT compared to mean pre-SCRT value of 97. LOTCA battery
used for patients aged 6 revealed respective average baseline, 6-month,
and 2 year follow-up values of 93, 102, and 98. Anxiety assessments with
C1, C2 (no anxiety 35) & HARS (normal 17) showed mean baseline
values of 39, 36, and 23 reducing to 29, 17, and 17 at 6-month and 23, 24,
and 14 at 2-year follow-up. Mean depression values using HDRS (normal
17) was 23 (range, 341) before and 14 after 2 years following SCRT.
For QOL in children, mean pre-SCRT HUI values of 9.5 improved to 8.8
at 2 years following SCRT. Barthels ADL were also maintained at follow-
up. Preliminary analysis of this relatively small cohort of young patients
treated with high-precision SCRT reveals maintained neuropsychological
prole assessed prospectively up to 2 years following treatment. However, it
clearly needs mature data in larger number of patients at a longer follow-up
to derive rm conclusions.
432. THE INCIDENCE OF CEREBROVASCULAR ACCIDENTS
(CVA) AND SECONDARY BRAIN TUMORS IN PATIENTS
WITH PITUITARY ADENOMA
P. Brown,
1
C. Brown,
1
R. Kline,
1
B. Pollock,
2
S. Stafford,
1
M. Link,
2
P.
Schomberg,
1
and R. Foote
1
;
1
Radiation Oncology and
2
Neurosurgery,
Mayo Clinic Rochester, Rochester, Minnesota, USA
The aim of this study was to assess the risk of CVA and secondary
brain tumors in patients with pituitary adenoma. A cohort of 144 patients
(median age 36.4 years; range, 12.386.8) from Olmsted County, Min-
nesota, diagnosed with pituitary adenoma between 1935 and 2000 was
studied. Only patients from Olmsted County were included because of the
unique nature of medical care in Olmsted County allowing the ascertain-
ment of virtually all cases of pituitary adenoma for this communitys resi-
dents during these years. Additionally, the patients with pituitary adenoma
represent a cohort from a much larger, well-studied population (i.e., the
entire population of Olmsted County), providing opportunities for future
comparisons. Seventy-seven patients (53.5%) underwent surgery for their
pituitary tumor, with 4 of these patients undergoing 2 operations. Twenty-
eight (19.4%) underwent radiation therapy (median dose 45 Gy; range,
17.660 Gy) with twenty of these patients (13.9%) undergoing both sur-
gery and radiation therapy (RT). Five patients received repeat RT (median
11.3 Gy; range, 8.450 Gy). Fifty-nine patients (41%) were simply observed
after their diagnosis of a pituitary tumor. Thirty -ve (24%) patients have
died with a median follow-up of 10.4 years (range, 0.153.1). The RT group
had signicantly (P 0.01) longer follow-up than the other patients, with
a median follow-up of 18.6 years versus 8.7 years, respectively. There were
no signicant differences in CVA rates between the 3 groups, observation,
surgery, and radiotherapy (10-year CVA rate 13.9%, 6.7%, 11.7%, respec-
tively). The combination of surgery and RT did not lead to an increased
risk of CVA when compared to surgery or RT alone. Analyzing only the
RT patients revealed a signicantly increased risk of CVA at 20 years for
patients after repeat RT (60%) compared to a single course of RT (22%,
P 0.05) but no difference with fraction sizes 2 Gy (P 0.24). Only
one secondary tumor was diagnosed, a meningioma 18 years after surgery
only for a prolactinoma. CVA is a signicant risk for patients with pituitary
tumors. Except for repeat RT, treatment does not seem to impact the risk.
Second malignancies in this population even with long-term follow-up are
a rare event. Future analyses will compare the rate of CVA in pituitary
patients to the general community.
433. SAFETY OF HIGH-PRECISION FOCAL CONFORMAL
RADIOTHERAPY EMPLOYING CONSERVATIVE MARGINS
IN CHILDHOOD BENIGN AND LOW- GRADE BRAIN
TUMORS
R. Jalali, B. Budrukkar, S. Sarin, and D. Sharma; Radiation Oncology,
Tata Memorial Hospital, Mumbai, India
The purpose of our study was to report safety of employing conserva-
tive margins in treatment planning and delivery of high-precision confor-
mal radiotherapy for childhood brain tumors. Between December 1999 to
December 2003, 31 children (22 boys and 9 girls, median age 12 years) with
incompletely excised or recurrent benign and low-grade brain tumors (14
craniopharyngiomas, 10 chiasmal/hypothalamic gliomas, 5 low-grade glio-
mas [LGG], and 2 others) were treated with three-dimensional conformal
radiotherapy (CRT) (12 patients) and stereotactic conformal radiotherapy
(SCRT) (19 patients). Gross tumor volume (GTV) included neuro-imaging
based visible tumor and/or resected tumor bed. Clinical target volume
(CTV) consisted of GTV 5 mm margin, and planning target volume
(PTV) consisted of additional 5 mm margin for CRT and 2 mm for SCRT.
Treatment was delivered with 3 to 9 conformal xed elds to a median dose
of 54 Gy/30 fractions. The actuarial 2-, 3-, and 4-year disease-free and
overall survival was 96%, 100%, and 100%, respectively (median follow-
up, 30 months; range, 1258 months). Radiological follow-up available in
30 patients revealed complete response in 1, partial regression in 12, stable
disease in 17, and progression in 1 patients (within the CTV). One patient
with craniopharyngioma on a routine imaging revealed a mild asymptom-
atic cyst enlargement, which resolved with conservative management. A
patient with chiasmatic glioma developed cystic degeneration and hydro-
cephalus 9 months after SCRT requiring cyst drainage and placement of a
VP shunt. Pre-irradiation evaluation showed hormonal dysfunction in at
least one endocrine axis in 16 patients. On follow-up, 2 out of the remain-
ing 15 patients also had hormonal impairment. Serial visual assessments
revealed impaired vision (acuity/elds) in 26 patients before starting RT,
which showed improvement in 16, stable in 14, and mild deterioration in 1
patient. Focal conformal radiotherapy techniques delivering irradiation to
a computer-generated target volume employing 7- to 10-mm 3D margins
beyond the visible tumor and/or resected tumor bed appear to be safe in
children with incompletely resected or recurrent benign/low-grade brain
tumors. Because of the ability of these techniques to achieve sharp dose
differential between the target volume and adjacent normal brain, long-
term prospective studies are required to test their potential objectively in
minimizing treatment related late morbidity and sustaining local control.
434. SPINAL RE-IRRADIATION AFTER SHORT- COURSE
RT WITH 1 8 GY OR 5 4 GY FOR METASTATIC SPINAL
CORD COMPRESSION
D. Rades,
1
L. Stalpers,
2
T. Veninga,
3
and P. Hoskin
4
;
1
Radiation
Oncology, University Hospital Hamburg-Eppendorf, Hamburg,
Germany;
2
Radiation Oncology, Academic Medical Center Amsterdam,
Amsterdam, The Netherlands;
3
Radiation Oncology, Dr. B. Verbeten
Institute Tilburg, Tilburg, The Netherlands;
4
Radiation Oncology,
Mount Vernon Cancer Centre, Northwood, UK
This study investigated the feasibility and the effectiveness of re-irradiation
(re-RT) for in-field recurrence of metastatic spinal cord compression
(MSCC) in long-term survivors (follow-up of at least 12 months), initially
treated with 1 8 Gy or 5 4 Gy. Of a total series of 540 MSCC patients,
who were irradiated with 1 8 Gy (n 261) or 5 4 Gy (n 279)
between 1/1995 and 8/2003, 56 patients (1 8 Gy, n 28, and 5 4 Gy,
n 28) could be identied who had a follow-up 12 months and were
treated with re-RT for in-eld recurrence of MSCC. Median follow-up in
these 56 patients was 17 (1258). Median time to recurrence was 6 (340)
months. Re-RT was performed with 1 8 Gy (after 1 8 Gy or 5 4 Gy,
n 32), 5 3 Gy (after 5 4 Gy, n 13), or 5 4 Gy (after 1 8 Gy,
n 11). The cumulative (primary RT plus re-RT) biologically effective dose
(BED) was 80 to 100 Gy2. Median follow-up after re-RT was 10 (423)
months. Motor function was evaluated up to 6 months after re-RT with a
5-point-scale (grade 0, normal strength; grade 1, ambulatory without aid;
grade 2, ambulatory with aid; grade 3, not ambulatory; grade 4, paraple-
gia). Twenty-four patients (43%) showed improvement of motor function,
29 (52%) no change, and 3 (5%) deterioration. Five of 6 previously non-
ambulatory patients regained the ability to walk. No second in-eld recur-
rence in the same spinal region was observed after re-RT. Outcome was
not signicantly inuenced by the radiation schedule. Radiation-induced
myelopathy was not observed. Spinal re-irradiation with 1 8 Gy, 5 3
Gy, or 5 4 Gy for in-eld recurrences of MSCC appears safe and effective.
Myelopathy seems unlikely, if the cumulative BED is 100 Gy2.
435. RADIOTHERAPY FOR MOTOR DEFICITS DUE TO
METASTATIC SPINAL CORD COMPRESSION: IS 1 8 GY AS
EFFECTIVE AS 10 3 GY?
D. Rades,
1
and L.J.A. Stalpers
2
;
1
Radiation Oncology, University
2
Radiation
Oncology, Academic Medical Center Amsterdam, Amsterdam, The
Netherlands
Since life expectancy is markedly reduced in patients with metastatic
spinal cord compression (MSCC), a short and effective radiation schedule
is desired. This study investigates a reduction of the overall treatment time
to only one day by comparing 1 8 Gy to the multi-fractionated 10 3 Gy
for functional outcome. Data of 204 patients, treated for MSCC with either
1 8 Gy (n 96) or 10 3 Gy (n 108), were analyzed retrospectively.
Motor function and ambulatory status were evaluated before and up to
24 weeks after RT. A multivariate analysis (nominal regression) was per-
formed, including radiation schedule, performance status, age, irradiated
vertebra, and the three relevant prognostic factors (type of primary tumor,
pre-RT ambulatory status, and time of developing motor decits prior to
RT). Improvement of motor decits was selected as basic category and com-
pared with no change. The univariate analysis showed no signicant differ-
ence between the two radiation schedules, either for improvement of motor
function (P 0.89 at 6 weeks after RT, P 0.89 at 12 weeks after RT, and
P 0.89 at 24 weeks after RT) or for post-treatment ambulatory rates (P
0.75 at 6 weeks after RT, P 0.52 at 12 weeks after RT, and P 0.67 at 24
weeks after RT). The multivariate analysis demonstrated a signicant effect
on functional outcome for the ECOG performance status (P 0.01), for the
pre-RT ambulatory status (P 0.01), for the type of primary tumor (P
0.01), and for the time of developing motor decits prior to RT (P 0.001),
but not for the radiation schedule (P 0.85). In the whole series, 23/68
(34%) initially non-ambulatory patients regained the ability to walk after
RT, 11/30 patients (37%) after 1 8 Gy, and 12/38 patients (32%) after
10 3 Gy (P 0.82). Our data suggest the two fractionation schedules to
be comparably effective for functional outcome. Thus, 1 8 Gy should be
considered for patients with a poor survival prognosis.
436. ROLE OF SURGICAL RESECTION IN COMBINATION
WITH FRACTIONATED STEREOTACTIC RADIOTHERAPY
FOR MANAGEMENT OF VESTIBULAR SCHWANNOMA
Y. Sawamura,
1
H. Shirato,
2
and T. Sakamoto
3
;
1
Neurosurgery,
2
Radiology, and
3
Otology, Hokkaido University Hospital, Sapporo,
Japan
The goal of this study was to clarify the role of surgery in combina-
tion with fractionated stereotactic radiotherapy (SRT) analyzing outcomes
in patients with vestibular schwannoma (VS). A total of 165 vestibular
schwannomas were treated with surgery and SRT. Twenty-four large
tumors of T4b (Hanover classication) were partially removed, and then
residual mass was treated by SRT. T4a and smaller tumors, 141 cases, were
treated with SRT alone at a radiation level of 40 to 50 Gy administered in
20 to 25 fractions. At the time of SRT, the median tumor size was 16.2
mm (range, 336 mm). The median follow-up period was 42 months. The
actuarial 7-year rate of tumor control (no growth 2 mm and no require-
ment for salvage surgery) was 91.8% (95% CI, 87%96%). Three patients
with progressive tumors underwent salvage tumor resection. All 24 tumors
that were partially removed before SRT were well controlled. The actuarial
5-year rate of hearing preservation (Gardner-Robertson Class IIV) was
71.5%. The observed complications of fractionated SRT included transient
facial nerve palsy (3% of patients), trigeminal neuropathy (9% of patients),
and balance disturbance (9% of patients). No new permanent facial weak-
ness occurred after fractionated SRT. Fractionated SRT resulted in an excel-
lent tumor control rate and produced a high rate of hearing preservation.
The role of surgery is only to reduce mass volume to the amount that can be
adequately treated by fractionated SRT. Only the large tumors of Hanover
Class T4b are the target of neurosurgical resection.
437. EFFECT OF MICROBEAM RADIATION EXPOSURE TO
THE MICROVASCULATURE OF HEALTHY MOUSE BRAIN
C. Rmy,
1
R. Serduc,
1
P. Vrant,
2
J. Vial,
2
R. Farion,
1
E. Brauer,
3
A.
Bravin,
3
J. Laissue,
4
C. Segebarth,
1
and B. van der Sanden
1
;
1
INSERM
U594 and
2
CNRS UMR 5588, Universit Joseph Fourier, Grenoble,
France;
3
ESRF, Grenoble, France;
4
University of Bern, Institute of
Pathology, Bern, Switzerland
One of the important side effects of brain tumor radiotherapy is the
breakdown of the blood-brain barrier (BBB), which may cause cerebral
edema and high intracranial pressure. A new technique, microbeam radia-
tion therapy (MRT) using synchrotron radiation X rays, has recently been
developed and is based on the idea that radiation damage in normal brain
tissue can be decreased by spatial microfractionation of the absorbed dose.
The aim of this study was to assess the early effects (2 h1 month) of MRT
on the microvasculature (BBB and blood volume) in the cortex of nude
mice using intravital two-photon microscopy. The upper part of the left
hemisphere of Swiss nude mice (5 weeks old) was irradiated in an antero-
posterior direction by a 3-mm-high, ~3.1-mm-wide array of 16 vertically
oriented, quasiparallel microplanar beams (width ~ 25 m, center-to-center
spacing ~ 207 m; entrance dose, 312 or 1000 Gy). At different time inter-
vals after MRT (2 h, 12 h, 24 h, 48 h, 4 d, 7 d, 12 d, 30 d), 3 mice were
anesthetized and placed on the motorized step stage of the two-photon
microscope after craniotomy (3 mm in diameter) and intravascular injec-
tion of 2 uorescent probes, FITC-dextran (70 kDa) and sulforhodamine
B (577 Da). The vascular volume in the irradiated portion of the brain was
estimated from z-scans at the FITC wavelength over a maximum distance
of 650 m starting from the dura. The vascular permeability was detected
as extravasations of sulforhodamine B in the irradiated microplanar tissue
slices. For all time intervals after MRT and both tested radiation doses,
the FITC-dextran remained in the functional vessels, and no signicant
change in vascular volume was observed. From 12 h until 12 days after
MRT with a 1000-Gy radiation entrance dose, diffusion of sulforhodamine
B in microbeam stripes was observed. No diffusion was detected one month
after MRT. It seems that a BBB breakdown occurs between 12 h and 12
days and is repaired between 12 and 30 days after irradiation. After 312
Gy, no leakage of sulforhodamine B in the microbeam stripes of normal
brain tissue was detected at any time after MRT. Up to one month after
a dose of 312 Gy applied in the microbeam mode, no radiation damage to
the microvasculature was detected in the irradiated microplanar slices of
normal brain tissue. This entrance dose would therefore be more appropri-
ate for the treatment of gliomas using crossred microbeams than a dose
of 1000 Gy.
438. SINGLE-FRACTION, IMAGE- GUIDED, INTENSITY-
MODULATED RADIATION THERAPY (IG IMRT) FOR
OLIGOMETASTATIC LESIONS OF THE SPINAL COLUMN
Y. Yoshiya,
1
M. Lovelock,
2
M.H. Bilsky,
2
M. Hunt,
2
and Z. Fuks
1
;
1
Radiation Oncology;
2
Medical Physics, Memorial Sloan Kettering
Cancer Center, New York, New York, USA
Stereotactic radiosurgery (SRS) utilizing high-dose, single-fraction
radiation has been shown to be very effective in the management of intra-
cranial metastases. Image-guided techniques were developed to deliver pre-
cise, high-dose, intensity-modulated radiotherapy (IMRT) in a single frac-
tion to metastatic lesions of the spinal column. Twenty-one oligometastatic
patients with spinal metastases near the spinal canal were immobilized in a
noninvasive cradle. IMRT was utilized to provide spinal cord dose-sparing
treatment plans (dmax 1000 cGy) while delivering 1800 to 2400 cGy
to the lesion (median, 2100 cGy) in a single fraction. Two-dimensional
and/or three-dimensional (cone beam CT) image-guided verication was
performed. Each patient was followed every three months with clinical and
radiographic (including MRI) assessment. No patient was lost to follow-up.
The noninvasive cradle coupled with IG IMRT provided set errors 1 mm.
The median maximum dose (dmax) to the gross tumor volume (GTV) and
spinal cord was 2640 cGy and 1042 cGy, respectively. The median average
cord dose was 434 cGy (range, 345768 cGy). With a median follow-up of 5
months (range, 315 months), 95% of patients have demonstrated durable
radiographic control and palliation of presenting symptoms. No signicant
treatment-related toxicity has been encountered, including myelopathy and
radiculopathy. High-dose, single-fraction IG IMRT is safe and effective.
Treatment precision approaches that expected from xed-frame SRS for
intracranial metastases, with similar rates of local control and minimal
toxicity.
439. COMPLICATIONS OF STEREOTACTIC
RADIOSURGERY IN PATIENTS WITH BRAIN METASTASES:
CONSIDERATIONS FOR DEEP AND FUNCTIONAL BRAIN
TUMOR LOCATIONS
D. Suki,
1
F.F.L. Lang,
1
M. Maldaun,
1
E.L. Chang,
2
and R. Sawaya
1
;
1
Neurosurgery and
2
Radiation Oncology, The University of Texas M.D.
Anderson Cancer Center, Houston, Texas, USA
Stereotactic radiosurgery (SRS) has become a common and popular
treatment for metastatic brain tumors over the past decade. The appeal of
SRS for the treatment of metastatic brain tumors stems from many factors
related to the inherent nature of the tumors themselves, as well as factors
related to the procedure. Among those factors is the widely held assumption
that SRS-associated morbidity is much less common than its neurosurgical
and other radiation counterparts, and that the procedure can be performed
in any brain location regardless of depth or regional brain function. We
performed a review of the published literature on SRS from 1990 to 2004 to
assess the adequacy of the evidence on the relationship between brain tumor
location and the risk of complications. We also reviewed our radiosurgery
experience among patients with metastatic brain tumors at M.D. Anderson,
correlating the risk of complications with deep and functional brain tumor
locations. The review of published literature failed to reveal adequate repre-
sentation of various brain tumor locations, including eloquent and/or deep
ones, or adequate description and analysis of the role of tumor location
as a predictor of SRS-related complications. Most published series either
do not mention location or provide the distribution of locations without
correlating these with complications. There is no report formally assessing
the threshold between maximum tolerated SRS doses and toxicity within
specic tumor locations. The review of the M.D. Anderson SRS history
showed that the majority of patients with lesions in eloquent areas expe-
rienced complications following the procedure. Complications were more
frequent in lesions located in eloquent brain areas (58% of 93 lesions) com-
pared to noneloquent areas (31% of 58 lesions) or near eloquent areas (39%
of 110 lesions) (P 0.05, log-rank test). Complications were also more
frequent in tumors located in the motor/sensory area or brain stem area
compared to others (57%, 65%, and 37%, respectively). The same trends
were seen when only severe complications (RTOG 3) were included, and
when failures, seizures, early deaths, headaches, nausea, and DVT were
excluded from the list of complications. Signicant multivariate predictors
of complications included eloquent tumor location, progressing primary,
and a melanoma primary cancer. The nding of a signicant independent
role of tumor location is especially compelling given the lower dose received
at some of these deep-seated locations. We propose a review of the notion
of safety of SRS in deep-seated and eloquent brain tumor locations based
on the following two premises: (1) the limited availability of published
data investigating the issue and (2) the recent data from our Center, which,
owing to longer and more complete follow-up, a detailed classication of
tumors by tumor functional location and grade, and adequate numbers in
various categories, support the hypothesis that the radiosurgical treatment
of metastatic brain tumors located in eloquent areas of the brain is associ-
ated with an increased risk of severe complications.
440. ANALYSIS OF HISTOPATHOLOGICAL AND
RADIOLOGICAL PARAMETERS TO PREDICT RESPONSE TO
RADIOTHERAPY IN LOW- AND HIGH-GRADE GLIOMAS
S. Al-Haddad,
1
D. du Plessis,
1
K. Kopitzky,
1
T. Smith,
2
D. Husband,
3

B. Haylock,
3
C. Walker,
3
and P. Warnke
1
;
1
Neurosciences, University
of Liverpool, Liverpool;
2
Radiology, Walton Centre of Neurology
and Neurosurgery, Liverpool;
3
Neurooncology, Clatterbridge Cancer
Research Trust, Wirral; UK
Limited information is available correlating radiographic response seen
after radiotherapy and survival outcome among patients with low- and
high-grade gliomas. There are no studies comparing radiographic response
between low- and high-grade tumors. The most widely used radiological
response measurement is based on the changes seen on contrast-enhanced
scans. However the majority of low-grade gliomas do not enhance, and
there is no established methodology applicable to both low- and high-grade
gliomas. The aim of this study was to analyze whether clinical parameters
or radiological changes observed after radiotherapy correlate with time
to progression (TTP) and overall survival (OS). Sixty adult patients with
WHO grade II and III supratentorial gliomas who had undergone stereotac-
tic or burr hole biopsy and were treated by primary radiotherapy between
1994 and 2000 were included in this analysis. Area and volume analy-
sis of both the contrast-enhanced regions and hypodense regions on CT
or hyperintense regions on T2 MR images was conducted by using NIH
Scion image analyzer to quantify the response in both low- and high-grade
tumors. Radiographic response to radiotherapy was correlated with time to
progression (TTP) and overall survival (OS). Of the 60 patients evaluated
for imaging response, the complete, partial response, stable disease, and
progressive disease rates were 5.0%, 26.7%, 43.3%, and 25% respectively.
Changes seen in hypodense area on CT or hyperintense regions on T2-
weighted image did not correlate with changes of the contrast-enhanced
regions. A signicant correlation between radiographic response and sur-
vival was observed (Cox regression, P 0.05). Grade II tumors showed
greater volume reduction after radiotherapy compared to grade III gliomas
(Mann-Whitney U test, P 0.05). Our ndings indicated that the presence
of contrast enhancement was signicantly associated with poorer prog-
nosis. Almost a third of low-grade and two thirds of high-grade gliomas
showed evidence of contrast enhancement. However, we found no evidence
to support the association between area or volume changes based on con-
trast-enhanced regions after radiotherapy with time to progression (TTP)
or overall survival (OS). Instead, the data gathered suggested that there was
a good correlation between volume changes seen on hypodense on CT or
hyperintense regions on T2-weighted image (CT/T2) with TTP and OS in
both low- and high-grade gliomas. Reduction in the volume of CT/T2 after
radiotherapy was signicantly associated with survival. The volume reduc-
tion was more evident in low-grade than high-grade gliomas. We found
that seizures at the time of presentation and radiographic response based
on Modied Macdonalds criteria to be independent prognostic factors
for time to progression. For overall survival, WHO histological grade and
radiographic response based on Modied Macdonalds criteria were found
to be independent prognostic factors. We recommend the use of volume
based on CT/T2 to standardize response measurement to treatment espe-
cially after radiotherapy.
441. PATTERN OF CARE AND SURVIVAL IN A
RETROSPECTIVE ANALYSIS OF 1866 PATIENTS (PTS) WITH
GLIAL TUMORS TREATED WITH RADIOTHERAPY (RT) IN
TWELVE ITALIAN CENTERS FROM 1985 TO 2003
M. Buglione,
1
R. Santoni,
2
L. Pirtoli,
3
V. Tombolini,
4
R. Bagnoli,
5

M.F. Osti,
6
L. Lupattelli,
7
A. Somensari,
8
U. Ricardi,
9
M. Krengli,
10

P. Giacobazzi,
11
and G.P. Biti
12
;
1
Radiation Oncology, University of
Brescia, Brescia;
2
Radiation Oncology, University Tor Vergata of Rome,
Rome;
3
Radiation Oncology, University of Siena, Siena;
4
Radiation
Oncology, University of LAquila, LAquila;
5
Radiation Oncology of
Arezzo, Arezzo;
6
Radiation Oncology, University La Sapienza of Rome,
Rome;
7
Radiation Oncology, University of Perugia, Perugia;
8
Radiation
Oncology of Cremona, Cremona;
9
Radiation Oncology, University of
Torino, Torino;
10
Radiation Oncology, University of Piemonte Orientale,
Novara;
11
Radiation Oncology of Modena, Modena;
12
Radiation
Oncology, University of Firenze, Firenze; Italy
The end points of our study were to analyze patterns of clinical pres-
entation, care, and outcome in a multi-institutional series of radiotherapy-
treated malignant glioma pts and to evaluate actuarial overall survival (OS)
in the different clinical and therapeutic subsets. Histology was reclassied
by using the WHO system; performance status was dened according to
the Karnofsky index and Order scale. Type of surgery, RT volumes, RT
techniques and doses, supportive care, and chemotherapy were analyzed
also according to the accrual period (19851990, 19911996, and 1997
2003). The OS was calculated only for the pts with G34 astrocytoma
(1466 pts), using the Kaplan-Meier method. Differences in actuarial overall
survival (OS) were analyzed with the log-rank test and the Cox regres-
sion test. Statistically signicant differences (0.000 P 0.02) in clinical,
diagnostic, and therapeutic features according to the accrual period are
evident. In the last period, many more pts were treated who were aged 60
years (27%, 42.3%, and 50.7%, respectively, in the 3 groups), with worse
Order score (78%, 66%, and 89%, respectively), with lesions 3 to 5 cm
large (35%, 45%, and 50%, respectively) with G4 disease (60%, 73%,
and 73%, respectively). As for the diagnostic workup, the number of pts
submitted to MRI or CT and MRI signicantly increase in the more recent
periods, both in the presurgical and in the postsurgical setting (P 0.000).
In the last period, more pts were submitted to radical surgery (P 0.037)
and to conformal radiotherapy (P 0.000), mainly on more limited vol-
umes (P 0.000). The majority of the pts were treated with RT doses 60
Gy (53.3%). Median OS of the entire series was 9 months. The univariate
analysis showed a better survival for young pts (P 0.0000), in those
with better Order score (P 0.0000), with G3 histology (P 0.0000),
and small disease (P 0.0027). Among treatment variables, radical sur-
gery (P 0.0001), high RT dose (P 0.0000), limited treatment volumes
(P 0.0000), and the use of chemotherapy (P 0.0000) were related with
a better survival. The multivariate analysis conrmed the importance of
histology, tumor size, age, neurological performance status, radical sur-
gery, dose of RT, and volumes of treatment. In Italy, patterns of practice
for malignant gliomas changed signicantly during the last two decades.
Staging procedures were increasingly accurate, surgery more aggressive,
and RT techniques more sophisticated. The relevance for OS of age, NPS
and WHO histology, radical surgery, high-dose radiotherapy on limited
volumes is conrmed.
442. DETECTION AND DIFFERENTIATION OF
LACTATE AND LIPIDS BY SINGLE VOXEL PROTON MR
SPECTROSCOPY
Y. Kajiwara, F. Yamasaki, T. Saito, K. Sugiyama, K. Arita, and K.
Kurisu; Department of Neurosurgery, Hiroshima University, School of
Medicine, Hiroshima, Japan
In single voxel proton MR spectroscopy (
1
HMRS), lactate and lipids
signals partially overlap, and it is sometimes difcult to differentiate lactate
and lipids signals in clinical settings. Our aim in this study is to identify
lactate and lipid by varying the TE, and we hypothesize that accurate detec-
tion of lactate and lipid has a powerful value in diagnosis of brain tumors.
Between August 1999 and February 2004,
1
HMRS was performed, and
meaningful spectra following in our protocol were obtained on 163 brain
tumor patients (101 men and 62 women; mean, 44.3 years old).
1
HMRS is
done with TE of 144 and then following TE of 30 and/or TE of 288 if neces-
sary. Of the 163 patients, the level of lactate was negative in 31 patients,
positive in 107 patients, and strongly positive in 25 patients. The level
of lipids was negative in 68 patients, positive in 45 patients, and strongly
positive in 50 patients. In glioneuronal tumors, lactate was detected in
54.5% of WHO grade 2 (positive 54.5%, strongly positive 0%), 93.1%
of grade 3 (positive 86.2%, strongly positive 6.9%), and 100% of grade 4
(positive 66.7%, strongly positive 33.3%). Lipids were detected in 3.0%
of WHO grade 2 (positive 3.0%, strongly positive 0%), 58.6% of grade 3
(positive 31.0%, strongly positive 27.6%), and 100% of grade 4 (positive
52.8%, strongly positive 47.2%). Detection rate of lactate was a signicant
factor for discriminating between WHO grade 2 and 3 (P 0.0239) and
grade 3 and 4 (P 0.0347). The detection rate of lipids was a more sig-
nicant factor to discriminate between WHO grade 2 and 3 (P 0.0073)
and between grade 3 and 4 (P 0.0048) in glioneuronal tumors. With our
varying TE method, it is possible to accurately and efciently detect lactate
and lipids in brain tumors. We showed a signicant correlation between
lactate/lipid expression and WHO grade in glioneuronal tumors.
443. QUANTIFICATION OF EARLY BRAIN TUMOR
THERAPEUTIC RESPONSE USING FUNCTIONAL
DIFFUSION MAPS
B.D. Ross,
1
B.A. Moffat,
1
T.L. Chenevert,
1
T.S. Lawrence,
2
C.R. Meyer,
1

T.D. Johnson,
3
L. Junk,
4
and A. Rehemtulla
2
;
1
Radiology,
2
Radiation
Oncology,
3
Biostatistics, and
4
Neurology, University of Michigan, Ann
Arbor, Michigan, USA
Assessment of the efcacy of radiation and chemotherapy for brain
cancer patients is traditionally accomplished by measuring changes in
tumor size several weeks after therapy has been administered. The abil-
ity to use noninvasive imaging during early stages of fractionated therapy
to determine if a particular treatment will be effective would provide an
opportunity to optimize individual patient management and avoid unneces-
sary systemic toxicity and expense. We investigated whether changes in the
Brownian motion of water within tumor tissue as quantied by diffusion
magnetic resonance imaging (MRI) could be used as an early, surrogate
marker for early prediction of treatment response in brain cancer patients.
Twenty brain tumor patients were examined by standard and diffusion
MRI before initiation of treatment. Additional images were acquired at
3 weeks post-initiation of chemo- and/or radio-therapy. Images were co-
registered to pretreatment scans, and changes in tumor water diffusion
values were calculated and displayed as a functional diffusion map (fDM)
for correlation with clinical response. Of the 20 patients imaged during
the course of therapy, 6 were classied as having a partial response (PR),
6 as stable disease (SD), and 8 as progressive disease (PD). FDMs were
found to predict patient response at 3 weeks from the start of treatment
revealing that early changes in tumor diffusion values could be used as a
prognostic indicator of subsequent volumetric tumor response. The use of
fDM provides an early surrogate marker for predicting treatment response
in brain tumor patients.
445. VALIDATION OF MOTOR PATHWAY TRACTOGRAPHY
BY COMPARISON WITH HEMIPARESIS IN PATIENTS
WITH INTRACRANIAL TUMORS
T.J.D. Byrnes, T.R. Barrick, B.A. Bell, and C.A. Clark; Clinical
Neurosciences, St. Georges Hospital Medical School, London, UK
Radiation necrosis is a dose-related consequence of radiotherapy and
necrosis within eloquent brain, including the motor pathways, causes per-
manent neurological decits. Conventional magnetic resonance imaging
gives limited information regarding the position of the white mater tracts,
especially when displaced by tumor. Diffusion tensor imaging (DTI) trac-
tography can demonstrate white matter tracts in vivo, and it has been
shown that incorporation of tractography into treatment planning can
reduce radiation dosage to white matter tracts, which may reduce the risks
of symptomatic radiation necrosis. Previously tractography required a large
amount of user interaction, prior knowledge, and time, placing it beyond
the scope of routine clinical practice. A new algorithm dramatically reduced
the time required to perform white matter tractography and allows motor
pathway segmentation with virtually no prior knowledge or interaction,
using a single standardized seed point. White matter tractography has been
validated by comparison with anatomical brain images, but there is a lack
of in vivo functional validation. We present the results from 25 patients
with intracranial tumors, 10 with a hemiparesis, comparing motor pathway
segmentation with motor function. Between March and October 2004, 25
patients, 18 male and 7 female, with an average age of 48 (2573) years,
underwent DTI prior to treatment for their supratentorial tumors. Tractog-
raphy was initiated from every voxel, and the geometry of the individual
bers from each voxel was computed. A single voxel was chosen bilaterally
within the anterior medulla. The geometry of the ber computed for that
voxel was compared with every other ber in the entire image and grouped
together if they were statistically similar, to segment the motor pathways.
There were 6 glioblastomas, 5 metastatic tumors, 6 grade 2 astrocytomas,
2 meningiomas, and 6 other tumor types. Ten patients had a clinically
demonstrable hemiparesis. All of those patients with a hemiparesis were
shown to have a clearly distorted and/or disrupted motor pathway. There
were 3 patients who appeared to have normal motor function but had a
reduction in the size of the motor pathways on tractography. This represents
100% sensitivity of motor tract disruption for hemiparesis with a specic-
ity of 80%. This study has demonstrated a semiautomatic segmentation
of the descending motor pathways with minimal user interaction or prior
anatomical knowledge, bringing the technique closer to being practical
within a clinical setting. We have shown that motor pathway disruption
is highly sensitive to hemiparesis, validating motor pathway segmentation
by tractography and clearly demonstrating its potential for radiotherapy
treatment planning.
446. IS THERE AN ASSOCIATION BETWEEN LOSS
OF HETEROZYGOSITY (LOH) ON CHROMOSOMES
1p/19q AND IMAGING FEATURES OF LOW- GRADE
OLIGODENDROGLIOMAS (LGO)?
S. Lieberman,
1
I. Lavon,
2
J. Gomori,
1
and T. Siegal
2
;
1
Neuroradiology
Unit, Department of Radiology;
2
Leselie and Michael Gafn Center for
Neuro-Oncology, Hadassah Hebrew University Hospital, Jerusalem,
Israel
LGO are diagnosed by their histologic features. Genetic analysis has
improved the distinction between subgroups of this tumor, and the com-
bined allelic loss on chromosomes 1p and 19q was found to be predic-
tive of favorable outcome. It is not known whether imaging features may
correlate with the biologic behavior of the tumor as predicted by current
chromosomal analysis. The objective of our study was to test the hypothesis
that genetic proles might be associated with typical imaging characteris-
tics identied in LGO. We retrospectively reviewed the MRI studies of 31
patients with histologically veried oligodendroglioma (WHO II) whose
median age was 39 years (range 2161). MRI images were assessed for the
following: rCBV grade, the presence of small cysts, the presence and relative
extent of non-cystic regions of FLAIR hypointensity which are hyperintense
on T
2
, for location and for
1
H-MRS ndings. A standard integral algorithm
calculated the peak areas of NAA, Cho, Cr, Lac, and myoinositol, and each
peak area was normalized for the Cr and the unsuppressed water peak.
LOH on chromosomes 1p and 19q was evaluated in paired tumor-blood
DNA samples, using PCR-based microsatellite analysis. LOH on chromo-
somes 1p/19q was found in 21 pts (68%) (group 1), and no deletions were
observed in 10 pts (32%) (group 2). Data on rCBV was available for 29
pts. High rCBV were detected in 10/20 pts (50%) in group 1 and in 6/9
pts (67%) in group 2 (P NS). Small cystic lesions were more common
in group 1 (67%) than in group 2 (40%), but it did not reached statistical
signicance. Hypointense FLAIR, hyperintense T2 foci engaged more than
50% of the tumor cut surface in 19% of group 1 patients and in 50% of
group 2 (P NS). Frontal lobe location was less frequent in group 1 (48%)
as compared with group 2 (7/10 pts [70%]), but it was not statistically
signicant. NAA, Cho, Cr, Lac and myoinositol ratios did not differ sig-
nicantly between the two groups. MRI characteristics cannot differentiate
reliably between subgroups of LGO when stratied according to their chro-
mosomal prole that frequently predicts chemosensitivity. Yet, high rCBV
was observed in 55% of pts with LGO, regardless of their 1p/19q status,
and was not associated with an unfavorable disease course. There is some
tendency for LGO with no loss on 1p/19q to have higher rCBV, less cystic
lesions, and more extensive manifestation of hypointense FLAIR. Still, this
requires a larger study to be veried.
447. PERFUSION-WEIGHTED IMAGING IN THE
ASSESSMENT OF BRAIN GLIOMAS
C. Chaskis, A. Michotte, T. Stadnik, K. Van Rompaey, and J. DHaens;
Neurosurgery, Academisch Ziekenhuis, Vrije Universiteit Brussel,
Brussels, Belgium
Because brain gliomas are histologically heterogeneous neoplasms,
biopsy targeting must be based on adequate identication of malignant
areas on imaging. Considering the reported differences in relative cerebral
blood volume (rCBV) among tumor grades, we evaluated whether preopera-
tive perfusion-weighted imaging (PWI) could improve the detection of areas
bearing a higher yield for malignancy. We studied a series of 55 consecutive
patients with newly diagnosed brain glioma. All patients underwent preop-
erative MR imaging and PWI. Surgery consisted of stereotactic biopsy in 29
cases and open craniotomy in 24 cases. Patients were followed up to 5 years
after the diagnosis to evaluate their nal survival. In our experience, PWI
added to standard MR imaging improved the choice of targets for biopsy
and the diagnostic accuracy, as conrmed by the clinical follow-up. Despite
a less precise demarcation of the tumor borders, it allowed a better local-
ization of its most malignant parts. Among the PWI parameters, we found
that only maximal rCBV ratios were clinically useful. PWI was a valuable
implement in the imaging assessment of brain gliomas, discriminating high-
from low-grade tumors. PWI correlated signicantly with the tumor grade
and the nal outcome (P 0.01).
448. CORRELATION OF HYPOXIC CELL FRACTION
WITH GLUCOSE METABOLIC RATE AND MARKERS OF
ANGIOGENESIS AND PROLIFERATION IN GLIOMAS
L. Cher,
1
C. Murone,
4
S. Ramdave,
2
A. Hannah,
2
G. OKeefe,
2

K. Pathmaraj,
2
H. Tochon-Danguy,
2
J. Sachinidis,
2
J. Chan,
2

S. Berlangieri,
2
G. Fabinyi,
3
and A. Scott
4
; Departments of
1
Oncology,
2
Nuclear Medicine and Centre for PET, and
3
Neurosurgery, Austin
Health, Parkville, Victoria;
4
Ludwig Institute for Cancer Research,
Tumor Targeting Program, Heidelberg, Victoria; Australia
In vitro studies suggest
18
F-uorodeoxyglucose (FDG) uptake in tumor
cells may reect both aerobic and anaerobic glycolysis.
18
F-Fluoromisoni-
dazole (FMISO) has been shown to selectively identify hypoxic but viable
tissue, which may contribute to chemoradiotherapy resistance in gliomas.
The aim of this study was to map and correlate tumor hypoxia with glucose
metabolic rate, angiogenesis, and proliferation in patients with low- and
high-grade gliomas. Seventeen patients with newly diagnosed primary brain
tumors were studied prospectively with
18
F-FMISO and
18
F-FDG positron
emission tomography (PET), and MP-RAGE MRI, prior to surgery. All
FMISO and FDG PET images were co-registered with the MRI for ste-
reotactic comparison of relative uptake. Biopsy samples at surgery were
histologically examined for expression of GLUT3, Ki67, HIF1a, VEGF,
VEGFR1, and microvessel density (MVD). In the 17 patients studied there
were 7 grade IV, 3 grade III, 4 grade II gliomas, and 2 metastatic adenocar-
cinoma.
18
F-FMISO uptake was found to correlate with tumor grade and
was often heterogeneous in the tumor. In high-grade gliomas
18
F-FMISO
uptake overlapped regions of maximal
18
F-FDG uptake. Low-grade glio-
mas uniformly did not take up
18
F-FMISO and were negative on
18
F-FDG
PET. GLUT3 expression correlated with
18
F-FDG uptake. Both
18
F-FMISO
and
18
F-FDG uptake was associated with a signicant increase in Ki67 and
VEGFR1 markers in tumor tissue (
18
F-FMISO, P 0.0001 and 0.0014;
and
18
F-FDG, P 0.00001 and 0.0012, respectively). There was a trend
for increased HIF1a expression with high
18
F-FMISO uptake. Expression
of HIF1a was associated with increased Ki67, VEGF, and VEGFR1 and
reduced MVD (P 0.05).
18
F-FMISO uptake correlates with glioma tumor
grade and
18
F-FDG uptake. Cellular maWrkers of hypoxia, proliferation,
and angiogenesis are strongly associated with hypoxic cell fraction and
glucose metabolic rate in gliomas. Further studies are planned to dene
the biology of hypoxia in high-grade gliomas and the predictive ability of
18
F-FMISO scans in glioma patients during therapy.
449. DIFFUSION-WEIGHTED MRI IN THE EARLY
DIAGNOSIS OF MALIGNANT GLIOMA
J. Baehring,
1
W. Bi,
2
and J. Piepmeier
2
;
1
Neurology and Neurosurgery
and
2
Neurosurgery, Yale University School of Medicine, New Haven,
Connecticut, USA
The diagnosis of glioblastoma multiforme is based upon imaging nd-
ings and histopathological features that are usually readily identied in
incisional or stereotacic biopsy specimens. A small subset of patients comes
to medical attention before the masses show rim enhancement and central
necrosis. As in those cases the tumors are typically located in eloquent areas
of the brain, tissue diagnosis is obtained through stereotactic biopsy and
not uncommonly is unsuccessful or does not allow for accurate grading. We
conducted this study in order to identify imaging characteristics of early
stages of malignant gliomas. This is a retrospective analysis of patients with
newly diagnosed malignant glioma seen on the Neuro-Oncology Service at
the Yale Brain Tumor Center between 2002 and 2004. Patients with typi-
cal radiographic presentation (large rim-enhancing masses on T1-weighted
MR images after administration of gadolinium) were excluded. Medical
records and magnetic resonance imaging studies of patients were reviewed.
Eight patients meeting the above inclusion criteria were identied. Diffu-
sion-weighted imaging (DWI) showed areas of increased signal intensity in
all cases. Low signal on apparent diffusion coefcient maps (ADC) indica-
tive of restriction of proton diffusion in corresponding areas was present
in six patients, whereas in the other two, ADC maps were not obtained.
In ve patients, patchy or small nodular enhancing lesions without central
necrosis were identied. Biopsy was performed in six patients at the time
of the above-described imaging ndings. Diagnosis of a malignant glioma
could only be established prior to further tumor growth in two cases. Glio-
blastoma can be a challenging diagnosis in a small subset of patients. Those
may particularly benet from early diagnosis and initiation of treatment,
as tumors are frequently located in a functionally important area in which
even minimal progression can give rise to substantial disability. Information
obtained through DWI and ADC maps should be incorporated in the clini-
cal decision-making process. For patients with masses displaying restricted
proton diffusion indicating high cellularity, even in the absence of contrast
enhancement, a biopsy should not be delayed.
450. PRIMARY CENTRAL NERVOUS SYSTEM
LYMPHOMA VOLUMETRIC ANALYSIS OF RESPONSE TO
CHEMOTHERAPY: IS THERE PREDICTIVE VALUE TO A
RESPONSE NOMOGRAM?
M.M. Mrugala,
1
S. Schreiber,
1
C.B. Knobbe,
1
A. Muzikansky,
3
G.J.
Harris,
2
J. Rabinov,
2
and F.H. Hochberg
1
;
1
Stephen E. and Catherine
Pappas Center for Neuro-Oncology,
2
Radiology, and
3
Biostatistics
Center, Massachusetts General Hospital and Harvard Medical School,
Boston, Massachusetts, USA
Primary central nervous system lymphoma (PCNSL) is a non-Hodgkins
B-cell lymphoma. Over 52% of patients achieve a complete response (CR)
following receipt of parenteral high-dose methotrexate (HD-MTX). We
provide HD-MTX (8 g/m
2
) every 14 days until a complete response (CR) is
achieved or disease progression is observed. Of importance to us is the sepa-
ration of early responder patients from those who will not achieve CR with
this therapy. We have been unable to use as predictors of CR the following
variables: age, performance status at diagnosis, or MTX pharmacokinetics.
The development of a response nomogram would provide early identi-
cation of patients likely to achieve a CR and to separate these individuals
from those with either progressive disease (PD) or partial response (PR)
for whom phase 1/2 trials would be most appropriate. We performed a
retrospective analysis of 20 patients whose PCNSL completely responded
to MTX in the absence of XRT or other chemotherapeutic agents. Ten male
and 10 female immunocompetent patients with median age at diagnosis of
64.5 years were included. All patients received MTX therapy, in the setting
of diminishing steroid dosing, for measurable disease (MRI-T1 gadolinium-
enhancing masses). Contrast-enhanced MRI scans were performed prior to
treatment and thereafter every 2 cycles. Tumor volumes (T1 gadolinium-
enhanced images and FLAIR images) were calculated by using Vitrea2 soft-
ware and a semi-automated edge detection system. The median baseline,
pretreatment, T1 post-gadolinium-enhancing tumor volume was 7.28 cm
3
,
and median baseline FLAIR volume was 64.49 cm
3
. At CR the enhancing
tumor volume was 0 and the median FLAIR volume was 29.52 cm
3
. Twenty
percent of patients achieved CR after 4 cycles, a further 15% after 3 cycles,
and an additional 15% after 5 cycles. Ten percent achieved CR following 6,
7, or 8 cycles. The remaining 5% of completely responsive patients required
9 to 12 cycles to achieve remission. The median time to achieve 50%, 75%,
and full reduction of the enhancing tumor volume from the start of therapy
was 1 cycle (14.5 days), 2 cycles (28 days), and 5.5 cycles (76 days), respec-
tively. We present (A) the relationship of baseline tumor volume to thera-
peutic response, (B) the relationship between response criteria based on
T1-gadolinium and FLAIR volumes, and (C) correlates of MRI volumetric
response including initial tumor volume, number of lesions, and patient age.
We conclude that construction of a nomogram, based on volumetric analy-
sis, can permit (A) rapid identication of non-responsive patients who will
be eligible for early-phase drug trials and (B) identication of populations of
MTX-responsive and non-responsive patients as the basis for pharmacoge-
nomic and chromosomal studies of drug response correlates.
451. SYSTEMATIC REVIEW OF THE DIAGNOSTIC
ACCURACY OF THALLIUM-201 SINGLE-PHOTON
EMISSION COMPUTED TOMOGRAPHY IN THE
DETECTION OF RECURRENT GLIOMA
M. Vos,
1
B. Tony,
2
O. Hoekstra,
2
T. Postma,
1
J. Heimans,
1
and L.
Hooft
3
;
1
Neurology,
2
Nuclear Medicine, and
3
Clinical Epidemiology and
Biostatistics, VU Medical Centre, Amsterdam, The Netherlands
The aim of this study was to determine the diagnostic accuracy of thal-
lium-201 single-photon emission computed tomography (201Tl SPECT) in
the detection of tumor recurrence in patients with previous radiotherapy for
supratentorial glioma. The databases of PubMed and Embase were searched
for relevant studies. Two reviewers independently selected and extracted
data on study characteristics, quality, and accuracy of studies. Studies were
included if they comprised at least six eligible patients, who underwent
201Tl SPECT (index test) and in whom (histo)pathological conrmation
(reference test) of the suspected brain lesion was obtained. Because of
the methodological and statistical heterogeneity of the included studies,
a quantitative meta-analysis was not performed. Instead, for every indi-
vidual study, the sensitivity, specicity, and diagnostic odds ratio (DOR)
of 201Tl SPECT was calculated. Eight studies met the inclusion criteria.
Only one study was considered of high methodological quality. Method-
ological limitations referred most notably to blinding and patient selection.
The DOR was greater than 1 in all included studies, with a broad range
(2.1350.9) and relatively wide 95% condence intervals. The sensitivity
of 201Tl SPECT ranged from 0.43 to 1.00, and the specicity from 0.25 to
1.00. We conclude that 201Tl SPECT is a valuable method in the detection
of tumor recurrence in patients treated with radiotherapy for supratentorial
glioma. However, the evidence is not very robust because of the low quality
and high heterogeneity of the studies included. Future studies are warranted
to further explore the diagnostic potential of 201Tl SPECT, and to deter-
mine optimum thresholds for the detection of glioma recurrence.
452. TAURINE DETECTED BY 1H MAGNETIC RESONANCE
SPECTROSCOPY IS INDICATIVE OF SUPRATENTORIAL
PRIMITIVE NEUROECTODERMAL TUMORS (PNETS)
H.G.J. Krouwer,
1,2
R.W. Prost,
3
W.M. Mueller,
2
G.P. Sinson,
2
C.J.
Schultz,
4
and K.-C. Ho
5
; Departments of
1
Neurology,
2
Neurosurgery,
3
Radiology (Neuroradiology),
4
Radiation Oncology, and
5
Pathology
(Neuropathology), Medical College of Wisconsin, Milwaukee,
Wisconsin, USA
Proton (1H) magnetic resonance spectroscopy (MRS) is a noninvasive
imaging technique to characterize intracranial lesions. With this modality,
medulloblastomas have recently been found to demonstrate elevated taurine
levels. Taurine is a non-proteinogenic amino acid present in high concen-
trations in fetal neural tissues and decreasing thereafter. We extend this
observation to include other primitive neuroectodermal tumors (PNETs)
with 2 cases of disseminated pineoblastomas. Spectra were acquired on
a 0.5 T clinical system with a quadrature transmit/receive head coil using
an elliptical excitation chemical shift imaging (CSI) sequence with TE
46 ms and TR 1000 ms. Taurine resonance was detected at 3.36 ppm.
This resonance becomes more detectable at 0.5T because of the collapse
of its complicated resonance in a manner similar to what has been shown
for glutamate (Prost et al., Magn. Reson. Med. 37, 615, 1997). Two male
patients, 32 and 47 years of age, presented with pineal region masses, both
with extensive leptomeningeal dissemination (4th ventricle and anterior
cervical spinal cord at C3-C4; auditory internal canals bilaterally, cerebel-
lar folia, along cervical and thoracic spinal cord and conus medullaris, as
well as all nerve roots) as assessed by conventional imaging studies. In both,
beta-HCG levels in CSF were slightly elevated, and AFP levels in CSF were
normal. MRS studies demonstrated, in addition to the resonances assigned
to myo-inositol, choline, creatine, N-acetylaspartate, glutamine/glutamate,
and lipid/lactate, a resonance at 3.36 ppm, compatible with taurine. Histo-
pathological specimens obtained from a drop metastasis at T10-T11, and
from the 4th ventricular mass, respectively, showed pineoblastoma. These
ndings support the hypothesis that detection of taurine by 1H-MRS in an
intracranial mass, supra- or infratentorial, may indicate a PNET histology.
Whether taurine as detected with the 0.5 T system is indeed specic for
PNETs requires further study.
453. COMPARISON OF GENOTYPE, HISTOPATHOLOGICAL
GROWTH PATTERNS, AND THE MR IMAGING
CHARACTERISTICS OF OLIGODENDROGLIAL NEOPLASMS
M. Jenkinson,
1
D. du Plessis,
1
E. Smith,
2
K. Joyce,
3
P. Warnke,
1
and C.
Walker
3
;
1
Neuroscience, University of Liverpool, Liverpool;
2
Neuro-
radiology, The Walton Centre for Neurology and Neurosurgery,
Liverpool;
3
Molecular Biology, Clatterbridge Cancer Research Trust,
Bebington; UK
Oligodendroglial neoplasms with loss of chromosomes 1p and 19q
are recognized to be more indolent with prolonged survival and increased
responsiveness to therapy compared with their morphologically equivalent
counterparts with intact 1p/19q. However, the biological basis of these
clinical differences is not known. Recent research suggests that genotype
inuences the magnetic resonance (MR) imaging characteristics of oligo-
dendroglial neoplasms, supporting the hypothesis that these tumors may
differ in their growth characteristics. The aim of this study was to examine
the histopathological growth pattern and MR imaging characteristics in
oligodendroglial neoplasms classied by genotype. Tumor imaging features
were assessed on T1 (gadolinium) and T2-weighted axial images: (1) sharp
versus indistinct border, (2) smooth versus irregular contour, (3) homog-
enous versus heterogenous signal intensity, and (4) contrast enhancement,
present versus absent. The histopathology of each case was assessed for
calcication and the growth pattern (solid, mixed, inltrative). In the 45
cases diagnosed with CT-guided serial stereotactic biopsy, the position of
each sample was recorded in relation to the biopsy tract, enabling char-
acterization of transitions in cellularity within the tumor margin. Allelic
imbalance in chromosomes 1p36 and 19q13 was determined. Eighteen
oligodendrogliomas (14 with 1p/19q loss) and 35 oligoastrocytomas (12
with 1p/19q loss) were investigated. Intact 1p/19q alleles correlated with a
sharp/smooth border in the whole group and grade II tumors (Fisher exact:
P = 0.037 and P = 0.007, respectively). Of the 7 cases with a sharp/smooth
border, 6 had intact 1p/19q. WHO grade III tumors were more likely to
have a solid growth pattern and WHO grade II tumors a mixed or inltra-
tive growth pattern (
2
: P = 0.001). Tumor genotype did not correlate with
growth pattern or transition in cellularity, but correlated with calcication
(Fisher exact: P = 0.031). Solid and mixed tumor growth patterns were
associated with contrast enhancement, and inltrative growth with no con-
trast enhancement (
2
: P = 0.008). All tumors with a homogenous signal
on T2 (n = 4) had a mixed growth pattern (
2
: P = 0.041). A sharp T1, but
not T2 tumor border was associated with sudden transition in cellularity
(Fisher exact: P = 0.048). In the absence of genetic testing, the observation
that oligodendroglial tumors with a sharp/smooth MRI border are more
likely to have intact 1p and 19q may be diagnostically useful. Growth pat-
tern, although related to contrast enhancement, was not associated with
MR tumor border appearance or genotype. These data suggest that growth
characteristics are unlikely to contribute to the differences in clinical behav-
ior of oligodendroglial neoplasms with or without loss of 1p and 19q.
454. IN VIVO MAGNETIC RESONANCE IMAGING OF
- GALACTOSIDASE IN A RAT C6 GLIOMA MODEL
S. Assadian,
1,2
B.J. Bedell,
2,3
S. Mzengeza,
2,4,5
A.C. Evans,
2,4,5
and R.F.
Del Maestro
1,4,5
;
1
Brain Tumor Research Center,
2
McConnell Brain
Imaging Center, and Departments of
3
Pathology,
4
Neurology, and
5
Neurosurgery, Montreal Neurological Institute, McGill University,
Montreal, Canada
With recent developments in tumor-targeted gene therapy, it has become
increasingly important to monitor the successful delivery of the gene to the
tumor. Current methods for determining reporter gene expression suffer
from serious limitations, including the invasive acquisition of tissue and the
inability to perform longitudinal studies on individual subjects. The poten-
tial to assess reporter gene expression using high-resolution, non-invasive,
in vivo imaging techniques would abrogate these limitations. As such, we
have developed a novel MRI probe which allows for in vivo measurement
of the expression of -galactosidase (-gal), which is commonly used as
a reporter of gene expression. Upon activation by tissue-localized -gal,
the probe increases the local blood-tumor permeability. Quantitative mea-
surement of the change in blood-tumor permeability, via the determina-
tion of extravasation of Gd-DTPA (Magnevist) into the tumor, provides an
estimate of relative -gal expression. In this study, we have evaluated the
ability of this probe to measure -gal expression in a rat intracerebral C6
glioma model in which tumor cells have been transfected to secrete -gal
into the extracellular space. C6 glioma cells were transfected in vitro with
the lacZ gene following an Igk secretion tag. Spheroids of the transfected
cell line were implanted into cerebral hemispheres of male Sprague-Dawley
rats. Animals implanted with non-transfected spheroids served as the con-
trol group. Two weeks post-implantation, the animals were imaged using a
Siemens 1.5 T whole-body MRI scanner. T1-weighted coronal images were
acquired following intravenous administration of probe and Gd-DTPA.
Following completion of imaging, the animals were sacriced and brains
snap-frozen in isopentane. X-gal staining was performed on cryostat sec-
tions to conrm the expression of -gal. MR data was processed using
software developed in-house to generate quantitative maps of -gal expres-
sion. Quantitative MR image maps successfully demonstrated the -gal
expression within the transfected tumors, but not the control tumors. The
secretion of -gal by the transfected tumor was conrmed by X-gal staining
of the corresponding cryosections. We have successfully imaged the expres-
sion of -galactosidase in vivo in a rat C6 glioma model using MRI. Our
probes will provide a powerful means for assessing the efcacy of in vivo
gene transfer and can also be manipulated to image the expression of other
proteins involved in targeted therapies of tumors.
455. CT-PERFUSION IN PRIMARY AND SECONDARY BRAIN
TUMORS: PRELIMINARY EXPERIENCE
A. Vidiri, A. Mirri, A. Fabi, S. Telera, A. Pace, M. Carosi, F. Portieri,
C. Carapella, and M. Crecco; Regina Elena National Cancer Institute,
Rome, Italy
CT-perfusion has been used to evaluate microvascular characteristics
of brain tumors and monitoring treatment effects. In the present study,
CT-perfusion has been performed with Multisections-CT (Volume Zoom
Siemens Medical Solutions); the images are post-processed in a dedicated
workstation, which yields the maximum intensity projection (MIP) images
and the maps of cerebral blood volume (CBV), cerebral blood ow (CBF),
and perfusion permeability (PTC). This technique has been presently used in
18 patients affected by brain metastases and 4 by malignant gliomas, mainly
with the goal of differentiating between radiation necrosis and tumor recur-
rence; the results have been related with FDG PET and SPECT scans and
subsequent surgical specimens. In some cases the tumors have been studied
before and after radiotherapy for dening tumor vascularization, and cor-
relating it with treatment-induced variations in tumor volume. Preliminary
data show good correlation between CT-perfusion and PET-SPECT results,
mainly in detecting the presence of radiation necrosis in metastatic lesions
submitted to stereotactic radiotherapy; in 3 patients affected by metastatic
lesions (two from breast cancer and one from melanoma) the CT-perfusion
showed the presence of the tumor in agreement with surgical reports; in
one case, CT-perfusion and SPECT allowed early documentation of relapse
in a patient affected by GBM who had had previous surgery and chemo-
radiation therapy. MR-perfusion is the gold standard in the evaluation of
brain vascularization; however, CT-perfusion represents an easy technique
with short acquisition and post-processing time. This technique is useful for
patients for whom MR is not suitable.
456. THE ROLE OF 111 INDIUM- OCTREOTIDE BRAIN
SCINTIGRAPHY IN THE DIAGNOSIS OF MENINGIOMAS
FROM OTHER CRANIAL BASED DURAL LESIONS
N. Nathoo,
1
K. Ugokwe,
1
A. Chang,
2
L. Li,
3
J. Ross,
2
J. Suh,
4

M. Vogelbaum,
1
and G. Barnett; Departments of
1
Neurosurgery,
2
Neuroradiology,
3
Biostatistics and Epidemiology, and
4
Radiation
Oncology, The Taussig Cancer Center and Brain Tumor Institute, The
Cleveland Clinic Foundation, Cleveland, Ohio, USA
Meningiomas are common extra-axial brain tumors with somatostatin
receptors that bind octreotide. We report the use of 111 indium-octreotide
brain scintigraphy (ObS) in the non-invasive differentiation of meningiomas
from other cranial dural-based pathology. A retrospective analysis of our
experience with ObS in the non-invasive differentiation of meningiomas
was performed. A neuroradiologist, blinded to the clinical data, used a stan-
dardized grading scheme to dene the uptake of octreotide and the pattern
of enhancement performed at 6 and 24 h postadministration. Correlation of
18
F-uoro-2-deoxy-d-glucose positron emission tomography (FDG-PET),
MRI scans, and octreotide uptake was performed. The cohort consisted of
49 patients (34 female; 15 males) with a mean age 62.4 years and a median
follow-up of 24 months. Management consisted of biopsy (n 3), resection
(n 10), observation (n 15), and radiation therapy (radiosurgery n
21; external beam n 3). ObS was correlated with FDG-PET brain studies
(n 37), histology (n 13), and angiography (n 1). The sensitivity,
specicity, accuracy, and positive predictor value for ObS were 100%,
60%, 85%, and 80%, respectively. The test successfully differentiated
meningiomas from a dural-based venous anomaly except for 2 false
patients (metastasis; chronic inammation). The addition of PET did not
improve the specicity of ObS to detect meningiomas. Correlation of ObS
vs. MRI (P 0.001), MRI vs. PET (P 0.0218), and octreotide vs. PET
(P 0.0071) revealed a signicant relationship. Octreotide scintigraphy
together with FDG-PET scanning increases the diagnostic specicity of
conventional neuroimaging when differentiating meningioma from other
dural-based pathology.
457. SENESCENCE, AUTOPHAGY, MORPHOLOGICAL
PLASTICITY, AND SURVIVAL OF GLIOMA CELLS EXPOSED
TO IONIZING RADIATION
W. Dos Santos, H. Fillmore, and W.C. Broaddus; Neurosurgery, Virginia
Commonwealth University, Richmond, Virginia, USA
Resistance to ionizing radiation (IR) and apoptosis is one of the key
factors contributing to the poor prognosis of patients with glioblastoma.
However, the mechanism underlying this resistance remains poorly under-
stood. Two approaches were used in this study to better identify relevant
events involved in radioresistance of glioma cells. First, glioma cells with
different p53 status (U87 wt, T98 mut, LN-Z308null) were distributed
into 100-mm plates, in triplicate. Cells were treated with single doses of IR
(6 Gy), administered ve consecutive times every six days. For each time,
samples were separated for protein extraction and further maintenance in
culture prior to analysis. A second approach aimed to select highly resistant
cells. Thus, cells were treated with high-dose radiation (60 Gy), adminis-
tered twice within a period of 6 days. Cells were analyzed for morpho-
logical changes by optical and electronic microscopy; proliferation rate and
viability were assessed by MTS assay and trypan blue; IR resistance was
monitored by clonogenic assay; changes in cell cycle were determined by
ow cytometry, and alterations in protein prole followed by Western blot
and immunocytochemistry. Antibodies to both p53 and p53-induced pro-
teins, as well as to differentiation markers and radiation resistant-related
proteins, were used. U87 glioma cells (wt p53) were more radioresistant
than T98G (mut p53). Exposure to consecutive doses of 6 Gy IR decreased
proliferation rate in a dose-dependent manner. However, with continuous
passage, irradiated cells were able to resume their normal rate of prolifera-
tion comparable to cells that did not receive IR. Resistant U87 cells exposed
to high-dose IR exhibited a striking change in morphology characterized by
increase in size (1015-fold larger than untreated control), increase in cyto-
plasm vacuolization that preceded the formation of neurite-like projections
and aneuploidy. These cells entered into a senescence state and survived
for at least one year with reduced proliferation. At the molecular level,
resistant U87 cell population showed an increased expression of p53 and
p21
cip/waf
protein, decreased expression of the undifferentiated marker nes-
tin, reduced levels of Rb protein but increased levels of survivin. These cells
also showed aberrant expression of the neuronal marker beta III tubulin in
addition to the astrocytic marker GFAP. In contrast, T98 cells showed no
alteration in the levels of these proteins but did not survive as long as U87.
In conclusion, our results suggest that radiation resistance may be in part
associated with the plasticity of some glioma cells to undergo senescence
and terminal differentiation followed autophagy. Furthermore, the p53 and
Rb pathway with participation of DNA repair and anti-apoptotic proteins
such as survivin are important players in radioresistance.
458. BIOLOGICAL EFFECTS OF PROTON BEAM
IRRADIATION ON GLIOMA CELL LINES IN VITRO
A. Gee,
1
A. Kacperek,
2
D. Spiller,
4
E. Shaw,
1
J. Dunn,
1
P. Warnke,
3

and C. Walker
1
;
1
Clatterbridge Cancer Research Trust, JK Douglas
Laboratory, Bebington;
2
Clatterbridge Centre for Oncology, Douglas
Cyclotron, Bebington; UK
Gliomas are considered to be one of the most radioresistant and least
curable tumors, and although many patients show an initial response to
radiotherapy, the majority recur within or adjacent to the target volume.
Proton therapy used in the treatment of some gliomas enables higher doses
of conformal irradiation to be given, while sparing critical normal tissue,
and offers slightly better linear energy transfer and radiobiological effects
than photons. However, cellular responses to protons have not yet been
extensively investigated. In this study, four glioma cell lines with differ-
ing molecular genetic characteristics were used to investigate the in vitro
biological effects of proton irradiation. Cells plated in monolayers at 5
10
4
cells per 35-mm dish were positioned in the modulated Bragg peak and
received 0, 2, 4, 6, 8, or 10 Gy proton irradiation (60 MeV) at a dose rate of
30 Gy min
-1
. Post-irradiation, Annexin-V and propidium iodide were added
to the media to assess apoptosis, and time-delay confocal microscopy was
performed over 65 h with an image recorded every 15 min. Flow cytomet-
ric analysis was performed 65 h post-irradiation. Monolayer cultures were
maintained post-irradiation to determine whether populations surviving
sequential fractions of proton irradiation could be established. T98G with
mutant p53 was the most resistant cell line, with little change in prolifera-
tion and apoptosis and cells accumulating in G
1
of the cell cycle follow-
ing 10 Gy irradiation. In contrast, U373, also with mutant p53, showed
complete inhibition of proliferation, and cells accumulated in G
2
M after
65 h. U-87 MG with wild-type p53 showed a similar cell cycle distribu-
tion and rate of apoptosis to the unirradiated control, yet proliferation
was inhibited. Hs683 ceased proliferating and demonstrated the greatest
level of apoptosis, with a 9-fold increase in Annexin-V staining at 65 h
post-irradiation, and surviving cells accumulated in G
1
of the cell cycle.
All cell lines produced populations that survived a single fraction of 10 Gy
protons. Surviving populations were established following 2 10 Gy for
U-87MG and 3 10 Gy for T98G. Expression proling using oligonucle-
otide microarrays to investigate differential gene expression in these cell
lines in response to irradiation is currently being undertaken. The four cell
lines with their different molecular genetic proles showed very different
responses to proton beam irradiation, suggesting that depending on their
genetic subtype, gliomas may display differential intrinsic radiosensitivity.
Through expression proling we aim to identify factors that contribute to
radiation resistance.
459. GROWTH DELAY OF HUMAN GLIOBLASTOMA
MULTIFORME SPHEROIDS AFTER THERMAL NEUTRON
IRRADIATION WITH GADOLINIUM (GDNCT) OR BORON
(BNCT)
R. Ciftci,
1
J.J. Hofstra,
1
F. Stecher-Rasmussen,
2
K. Franken,
1
R.
Kuperus,
2
R. Huiskamp,
2
J. Haveman,
1
and L. Stalpers
1
;
1
Radiotherapy,
Academic Medical Center, Amsterdam;
2
Nuclear Research Group
(NRG), Petten; The Netherlands
The survival of patients with GBM continues to be dismal despite
various modalities of treatment. Neutron capture therapy (NCT) is an
experimental treatment modality: Following thermal neutron irradiation
of nuclear elements with a high propensity to capture neutrons, such as
boron-10 (
10
B) or gadolinium-157 (
157
Gd), a high dose of secondary radia-
tion is released according to the following nuclear reactions.
157
Gd nth
158
Gd g e
-
Q 7.94 MeV
10
B nth
4
He
7
Li g Q 2.79 MeV
Certain compounds, such as boron phenylalanine fructose (BPA-F) and
Gd-DTPA, can accumulate locally in a brain tumor. Neutron irradiation
of a patient receiving boron (BNCT) or gadolinium (GdNCT) may reach a
tumoricidal local irradiation. In this study we investigated if the theoretical
effectiveness of BNCT and GdNCT works in the in vitro GBM tumor-
spheroid model. Multicellular spheroids of the human GBM cell line Gli-6
were cultivated up to a diameter of about 300 microns. Clinically achievable
concentrations of gadolinium-DTPA (Magnevist; 2.5 mmol) and BPA-F (30
ppm) were used as neutron capture agents. The spheroids were irradiated
with thermal neutrons in a dose of 0.3 Gy and 0.6 Gy at the Low Flux Reac-
tor (NRG, Petten) or with cesium-137 X rays (photons) up to 8 Gy (AMC,
Amsterdam). The size of the spheroids was measured for some weeks.
Irradiation of spheroids with X-ray-photons shows a dose-dependant
growth delay; Gd-DTPA or BPA-F did not alter the tumor response to X
rays. Irradiation of spheroids with thermal neutrons gives a dose-dependant
growth delay. The radiobiological effectiveness (RBE) of thermal neutrons
is about 5- to 10-fold that of X-ray photons; combination with Gd-DTPA
or BPA-F caused a signicant prolonged growth delay. The prolongation
is somewhat more pronounced in Gd-DTPA treated spheroids, both after
0.3 Gy neutron irradiation, and more pronounced after 0.6 Gy neutron
irradiation. Both Gd and B, at a clinically feasible concentration, produce
a strong growth delay of GBM spheroids after neutron irradiation. This
radiosensitization by Gd and B was, as expected, not observed after sphe-
roids to photon irradiation. The NCT-effect of Gd cannot be explained by
the release of secondary Auger electrons (as these do not penetrate more
than 1 or 2 cell layers), and should be attributed to the secondary release of
longer range photons and nuclear conversion electrons.
460. ABSCOPAL EFFECT ON SUBCUTANEOUSLY
IMPLANTED N29 RAT GLIOMA TUMORS, FROM
CONTRALATERAL TREATMENTS WITH PULSED
ELECTRIC FIELDS, RADIATION THERAPY, AND
IMMUNIZATION WITH SYNGENEIC INTERFERON-
GAMMA-SECRETING TUMOR CELLS
B.R.R. Persson,
1
C. Baurus Koch,
1
G. Grafstrm,
1
C. Ceberg,
1
B.
Widegren,
2
and L.G. Salford
3
; Departments of
1
Radiation Physics,
2
Tumor Immunology, and
3
Neurosurgery, Rausing Laboratory, Lund
University, Lund, Sweden
Effects of radiation therapy on cancer tumors outside the radiation eld
are a phenomenon known as the abscopal effect. The aim of the present
study is to study the effect of immunization with syngeneic interferon-
gamma (IFNg)-secreting cells on abscopal regression of contralateral, sub-
cutaneously implanted rat glioma tumors treated with radiation therapy in
combination with pulsed electric eld treatment. The study was performed
on rats of the Fischer-344 strain with rat glioma N29 tumors implanted
subcutaneously on the ank or on both the right treated hind leg and the
left untreated hind leg. Pulsed electric eld with 16 exponentially decaying
pulses with a maximum electric elds strength of 1300 V/cm and t
1/e
1
ms were rst applied to the tumors. Then within 5 min radiation treatment
was given in daily fractions of 5 Gy, to a total absorbed dose of 20 Gy. The
animals were arranged into one group of controls and 3 groups of various
treatments: pulsed electric elds only (PEF), radiation therapy (RT) with
60
Co-gamma radiation only (5 Gy per session), or combination of PEF and
RT. Tumors were treated 4 consecutive days at about 4 weeks after inocu-
lation, when a solid tumor has grown to a diameter of 1 to 1.5 cm. Once
weekly for three weeks, the animals were given intraperitoneal injections of
irradiated, modied N29 tumor cells, secreting IFN-g. The abscopal effect
was evaluated by comparing the growth rate of the untreated contralateral
tumors with the treated tumors. Fitting the data obtained from consecutive
measurements of tumor volume (TV) of each individual tumor to an expo-
nential model TV = TV
0
exp[TGR t] estimated the tumor growth rate
(TGR %per day) after the day of treatment (t 0). In total, 5 experimen-
tal series were performed with total number of 141 rats, of which 45 were
controls and the number of animals in each treatment group was around
30. The TGR of both types of tumors treated with the combination of PEF
and RT are signicantly decreased compared to the controls. The tumor
growth rate for both types of tumors is signicantly decreased by combined
treatment with PEF and RT compared to RT alone. There was no signicant
difference in contralateral tumor growth in animals without treatment and
those treated with IFN-g-secreting cells only. But a signicantly decreased
growth rate of the untreated tumor was observed in animals given PEF and
RT combined with IFN-g-secreting cells. These results indicate that the
most pronounced abscopal effect was achieved using pulsed electric elds
and radiation therapy combined with immunization using syngeneic tumor
cells.
461. CORRELATIVE MR IMAGING WITH GBM CANCER
GENOMICS
M. Kuo,
1
C. Nardini,
2
S. Cha,
3
and M. Diehn
4
;
1
Radiology, University
of California San Diego, San Diego, California, USA;
2
Computer
Science, University of Bologna, Bologna, Italy;
3
Radiology, University
of California San Francisco, San Francisco, California, USA;
4
Medicine,
Stanford University, Stanford, USA
Glioblastoma multiforme (GBM) is characterized by genetic instabil-
ity and an extremely variable appearance on magnetic resonance imaging
(MRI). We sought to determine whether characteristic GBM imaging den-
ing features seen on MRI could capture signicant alterations in intrinsic
gene expression signatures identied by gene expression microarrays. Four
imaging parameters were dened a priori to reect fundamental GBM-
dening MR imaging characteristics (degree of contrast enhancement,
necrosis, T2 heterogeneity, mass effect). MR images (T1, T2, contrast
enhanced) of 20 GBMs in 20 patients were evaluated and scored across
these 4 parameters by 2 radiologists in consensus and without knowledge
of the matching GBM genomic information. Unsupervised and supervised
analyses were performed for correlation of the imaging parameters against
the corresponding matched GBM microarray data (each microarray con-
taining 23,000 clones) to identify relationships between imaging traits and
tumor gene expression. Four of four imaging traits demonstrated statisti-
cally signicant correlations with large-scale alterations in gene expres-
sion: degree of contrast enhancement (P 0.01), necrosis (P 0.01), T2
heterogeneity (P 0.01), and mass effect (P 0.005). Further, there was
signicant enrichment of genes identied by 3/4 imaging traits in several
fundamental GBM gene expression signatures: Degree of contrast enhance-
ment was enriched for the hypoxia/angiogenesis (P 3.9 10
43
), extra-
cellular matrix (P 1.6 10
48
), and immune inltration (P 9.1
10
-56
) signatures, and necrosis and mass effect were both enriched for the
proliferation signature (P 1.6 10
14
and P 3.2 10
51
, respectively).
Radiogenomic evaluation of GBM on MRI can potentially co-localize sev-
eral characteristic imaging ndings to fundamental GBM gene expression
signatures and may provide plausible insights into GBM tumor biology
based on imaging alone.
462. IMAGING PRIMARY BRAIN TUMORS WITH [18F]-
3-FLUORO-AMINOCYCLOBUTANE CARBOXYLIC ACID
[FACBC] PET: A PILOT STUDY
A. Demopoulos, T. Akurst, B. Beattie, J. Pena-Gomez, H. Schoder, N.
Pillarsetty, S. Larson, R. Finn, and R. Blasberg; Neurology, Memorial
Sloan Kettering Cancer Center, New York, New York, USA
In the management of primary brain tumors, new or expanding lesions
observed on MRI may represent recurrent tumor, treatment effect, or necro-
sis.
11
C-methyl-methionine (
11
C-MET) PET imaging has several advantages
over [18F]-uoro-2-deoxyglucose PET, but is confounded by the presence of
radiolabeled metabolites. Logistically,
11
C is more challenging because of a
short half life (20 min vs. 110 min for
18
F).
11
C requires on-site production
and is less widely available. For these reasons and substantial cost savings,
3-uoro-aminocyclobutane carboxylic acid (18F-FACBC) PET studies may
be superior to both [18F]-uoro-2-deoxyglucose and
11
C-MET PET. We
report interim results of a pilot study evaluating dosimetry and imaging
characteristics of
18
F-FACBC in comparison to
11
C-MET PET. Patients with
previously treated primary brain tumors and a suspicion for recurrent or
progressive disease were included. All patients had
18F
-FACBC,
11
C-MET
PET, and recent MRI with gadolinium. Because of the shorter half-life,
11
C-
MET PET imaging was performed rst, followed by
18
F-FACBC imaging 2
h later.
18
F-FACBC brain and body dosimetry information were collected at
t 0 (brain only), 1, 2, and 3 h. Blood and urine sampling was performed
for radioactivity concentration and metabolite analysis via HPLC. MRI
coregistration was performed in all patients. Descriptive SUVmax statistics
were recorded and compared with tissue pathology and clinical outcome,
where available. In this small series, we found good concordance between
11
C-MET and
18
F-FACBC PET imaging. Isotope localized to tumor as visu-
alized on MRI. Uptake of
18
F-FACBC was delayed in non-contrast enhanc-
ing regions. No metabolites were detected in blood or urine. In conclusion,
18
F-FACBC PET imaging is a promising and cost-effective imaging modality
for primary brain tumors. Larger studies will further delineate its role in the
management of these difcult neoplasms.
463. IMAGING CORRELATES OF MOLECULAR SIGNATURES
IN OLIGOASTROCYTOMAS
M. Eoli, C. Maccagnano, L. Bissola, A. Silvani, B. Pollo, T. De Simone,
D. Bianchessi, A. Boiardi, G. Finocchiaro, and M. Bruzzone; Clinical
Neuro-Oncology, Neurological Institute C. Besta, Milan, Italy
In order to improve therapeutic management of oligoastrocytomas
(OA) we investigated the relationship between magnetic resonance imaging
(MRI) features on the pre-operatory scan, the histopathological diagnosis,
and the genetic signatures of these tumors. Sixty-two gliomas were classi-
ed by histological analysis as grade II (n 31) or grade III OA (anaplastic
OA, AnOA; n 31). Loss of heterozygosity (LOH) with different microsat-
ellite markers was studied on chromosomes 1p, 10q, 17p, and 19q. On each
MRI examination the following parameters were analyzed: location of the
tumor, sharpness of tumor border on T1-weighted images, homogeneity of
the tumor signal on T1 and T2 weighted images, mass effect and presence
of contrast enhancement. In a series of 62 cases we observed that 1p and/
or 19q LOH correlates with homogeneous T1 and T2 appearance (25/38,
P 0.008) and with decreased frequency of temporal location (3/38, P
0.002). Indistinct border, lack of T1 and T2 homogeneity, and presence of
enhancement were signicantly associated with LOH on 10q (P 0.001).
These ndings indicate that molecular alterations associated with cancer
may confer physical or biochemical characteristics to the tumor that can be
imaged. In oligoastrocytomas, LOH on 10q, a marker of astrocytoma pro-
gression, correlates well with MRI ndings of high-grade gliomas (indis-
tinct border, T1 and T2 lack of homogeneity, and presence of enhancement).
Genotypic assessment, in addition to MRI and histological evaluation, may
improve the management of patients with oligoastrocytomas.
464. IMAGING TUMOR PROLIFERATION: VALIDATION OF
3-DEOXY-3-[F-18]FLUOROTHYMIDINE (FLT) POSITRON
EMISSION TOMOGRAPHY IN CEREBRAL GLIOMAS
S. Price,
1
D. Wang,
2
T. Fryer,
2
A. Dean,
5
P. Hutchinson,
1
N. Burnet,
3

J. Pickard,
1
and J. Gillard
4
;
1
Academic Neurosurgery Unit,
2
Wolfson
Brain Imaging Centre, and Departments of
3
Oncology and
4
Radiology,
University of Cambridge, Cambridge;
5
Department of Neuropathology,
Addenbrookes Hospital; Cambridge; UK
Uncontrolled cellular proliferation is one of the cardinal features of
malignant tumors. 3-Deoxy-3-[F-18]uorothymidine (FLT) is a compound
that is taken up into dividing cells and phosphorylated by thymidine kinase
1 (TK1), which leads to intracellular trapping. FLT activity is therefore a
measure of cellular TK activity and hence cellular proliferation. Its location
within the body can also be imaged by using positron emission tomography
(PET). Twelve patients with cerebral gliomas (mean age, 49.6; 4 WHO
grade IV, 4 WHO grade III, and 4 WHO grade II tumors) were studied pre-
operatively; 200 MBq of FLT was given intravenously and a 3D dynamic
PET study performed. Venous blood sampling was performed throughout
the study. Maps of FLT uptake were generated and coregistered to an MR
study used for biopsy planning. All patients underwent an image-guided
biopsy, with biopsies taken at intervals along the biopsy tract. Biopsies were
parafn embedded and sections used for regular histopathological analysis
and immunohistochemistry using the MIB-1 antibody as a marker for cel-
lular proliferation. The coordinates for all biopsy sites were determined,
and uptake values for FLT were determined. There was little uptake of FLT
in either the normal brain or the low-grade gliomas, except in one case
where the MIB-1 labeling index was 12% without features of anaplastic
transformation. In 3 of the WHO grade III tumors the MRI showed little
enhancement, but there was focal increase in FLT in these cases. In all grade
IV tumors there was marked uptake of FLT. The maximum FLT uptake
from all biopsy regions correlated with the maximum MIB-1 labeling index
(Pearsons correlation coefcient r 0.69; P 0.03). By using a threshold
of 1.5 10
3
ml
plasma
min
-1
ml
brain
-1
) for the individual biopsy sites, FLT could
detect tumor with a sensitivity of 92% and a specicity of 64%; it was mar-
ginally more sensitive but less specic than either T
2
- or contrast-enhanced
T
1
-weighted MRI. FLT is a good imaging marker of tumor proliferation.
It may be especially useful in monitoring response to therapy and guiding
image-guided biopsies in minimally enhancing tumors.
465. ADVANCES IN MANAGEMENT OF MALIGNANT
MENINGITIS
M.C. Chamberlain; University of California, Norris Cancer Center,
Los Angeles, California, USA
Neoplastic meningitis (NM) is a common problem in neuro-oncology,
occurring in approximately 5% of all patients with cancer, and is the third
most common site of central nervous system (CNS) metastases. NM is a dis-
ease affecting the entire neuraxis, and therefore clinical manifestations are
pleomorphic, affecting the spine, cranial nerves, and cerebral hemispheres.
Because of craniospinal disease involvement, staging and treatment needs
encompass all cerebrospinal uid (CSF) compartments. Treatment of NM
utilizes involved-eld radiotherapy of bulky or symptomatic disease sites
and intra-CSF drug therapy. The inclusion of concomitant systemic therapy
may benet patients with NM and may obviate the need for intra-CSF che-
motherapy. At present, intra-CSF drug therapy is conned to three chemo-
therapeutic agents (i.e., methotrexate, cytosine arabinoside, and thio-TEPA)
administered by a variety of schedules either by intralumbar or intraventric-
ular drug delivery. Although treatment of NM is palliative, with an expected
median patient survival of 2 to 6 months, it often affords stabilization and
protection from further neurologic deterioration in patients with NM.
466. ADVANCES IN MANAGEMENT OF BRAIN METASTASIS
R. Sofetti, R. Rud, E. Laguzzi, and E. Trevisan; University of Torino,
Neuroscience, Torino, Italy
The management of patients with brain metastases has improved over
time, because of advances in technology and a better knowledge of prog-
nostic factors, leading to a more accurate patient selection. In the diagnostic
setting, MRI is the gold standard, and MRI diffusion/perfusion and MRS in
certain circumstances improve the diagnostic accuracy. A tissue diagnosis is
needed in patients with unknown primary tumor or with absent/controlled
systemic disease when a long interval has elapsed since the initial cancer
diagnosis or with active but treatable systemic disease when the radiologic
appearance is atypical. Whole-body FdG PET is a sensitive tool to detect
pulmonary foci as probable primary tumors in patients with biopsy-proven
brain metastasis and a negative cancer history. Neurocognitive tests are a
relatively sensitive measure of brain functioning, being important in pre-
dicting survival and monitoring treatment effects and tumor progression.
The combination of surgical resection and WBRT is superior to WBRT
alone for the treatment of single brain metastasis in patients with limited or
absent systemic disease and good performance status. Radiosurgery yields
results that are comparable to those reported after surgery, provided that
the lesions diameter does not exceed 3 to 3.5 cm. Radiosurgery combined
with WBRT (radiosurgical boost) is superior to WBRT alone in single but
not in multiple brain metastases. Hypofractionated stereotactic radiother-
apy can be an alternative to radiosurgery. Still controversial is the need for
WBRT after surgery or radiosurgery: Local control on MRI is signicantly
better after the combined approach, but overall survival does not improve.
Phase 3 studies (RTOG, EORTC, etc.) are ongoing, trying to identify which
subgroups of patients are candidates for WBRT after local treatments. A
new form of brachytherapy (Gliasite Radiation Therapy System) is now
being investigated in patients who have undergone surgical resection. Novel
radiation sensitizers (Motexan gadolinium, RSR 13) have some activity
in conjunction with WBRT. The response rate of brain metastases to che-
motherapy is similar to that of the primary tumor and extracranial metas-
tases. Promising results have been recently reported with temozolomide
in melanoma, capecitabine in breast cancer, and getinib (ZD 1839), an
EGFR tyrosine kinase inhibitor, in NSCLC. Chemotherapy may represent
the initial treatment (with or without subsequent WBRT) in patients with
multiple brain metastases from NSCLC and breast cancer who are asymp-
tomatic, whereas WBRT remains the treatment of choice in symptomatic
patients. Cognitive defects in long-surviving patients after WBRT are a real
concern: Acetylcholinesterase inhibitors (donepezil, memantine) could be
of some efcacy in improving cognitive functioning.
468. NOA- 03 MULTICENTER TRIAL OF HIGH-DOSE
METHOTREXATE IN PRIMARY CNS LYMPHOMA: FINAL
REPORT
U. Herrlinger,
1
W. Kker,
2
M. Uhl,
1
H.P. Blaicher,
3
H.-O. Karnath,
3

L. Kanz,
4
M. Bamberg,
5
and M. Weller
1
; Departments of
1
General
Neurology,
2
Neuroradiology,
3
Cognitive Neurology,
4
Medical Oncology,
and
5
Radiation Oncology, University of Tbingen, Tbingen, Germany
The German multicenter NOA-03 trial explored the value of primary
high-dose methotrexate therapy (HD-MTX; 8 g/m
2
) alone in patients with
primary CNS lymphoma (PCNSL). Response data have already been pub-
lished (Herrlinger et al., Ann. Neurol. 51, 247, 2002). The present report
summarizes the long-term outcome and the development of late neurotoxic
sequelae in the NOA-03 trial. Patients were evaluated for progression-free
and overall survival. In patients surviving more than 12 months, the devel-
opment of leukoencephalopathy on MRI was analyzed as a marker for late
neurotoxicity. Six long-term survivors were tested for decits in cognitive
function and for quality of life using the EORTC-QLQ C30 and Brain
20 questionnaires. Thirty-seven patients were enrolled in the trial. Eleven
patients achieved a complete remission (CR) with HD-MTX. Ten of these
patients relapsed after a median time of 15 months (246 months), 4 of
them with a systemic relapse. Thirty-three patients received second-line
therapy. Two of 7 (28%) patients receiving PCV (procarbazine, lomustine,
vincristine) and 12 of 20 patients (60%) receiving whole-brain radiotherapy
(WBRT) achieved a CR upon second-line therapy. The median survival
after the start of second-line therapy was 19 months. There was no signi-
cant difference between patients receiving WBRT (19 months) and PCV (12
months). The overall median survival was 25 months with a 2-year survival
rate of 51% and a 3-year survival rate of 29%. Four years after the start of
therapy, 34% of patients surviving more than 12 months had developed leu-
koencephalopathy. The rate of leukoencephalopathy was higher in patients
treated with WBRT (58%) than in patients treated without WBRT (10%).
Neuropsychological testing revealed mild to extensive attention decits in
all 6 patients tested (5 without WBRT, 1 with WBRT) and memory decits
in 4 of 6 patients. Two patients had normal, 3 had moderately restricted
quality of life, and the only patient tested who had received WBRT had
markedly restricted quality of life. High-dose MTX therapy with deferred
radiotherapy had moderate efcacy. Radiotherapy was an effective second-
line therapy but may induce a higher rate of late neurotoxicity. The nal
results of the NOA-03 trial illustrate the need for more effective rst-line
treatments.
469. TEMOZOLOMIDE AS PRIMARY CHEMOTHERAPY
FOR LOW- GRADE OLIGODENDROGLIAL TUMORS (LGOT):
PREDICTIVE IMPACT OF CHROMOSOME 1P LOSS ON
RADIOGRAPHIC RESPONSE
K. Hoang-Xuan,
1
E. Crinire,
1
J. Lejeune,
1
F. Diakite,
1
S. Taillibert,
1

F. Laigle-Donadey,
1
L. Capelle,
2
A. Carpentier,
1
M. Sanson,
1
and
J. Delattre
1
; Department of
1
Neurology & INSERM U495 and
2
Department of Neurosurgery, CHU Piti-Salptrire, Paris; France
Temozolomide (TMZ) has recently been shown to be active as initial
treatment for low-grade oligodendroglial tumors (LGOT). Preliminary
results suggested that chromosome 1p loss may predict the response to
TMZ in LGOT (Hoang-Xuan et al., J. Clin. Oncol. 22, 3133, 2004). The
objectives of our study were to conrm in a larger series and with a longer
follow-up (1) the efcacy of TMZ on LGOT and (2) the predictive value of
1p loss on the radiographic response. Patients suffering from WHO grade
II oligodendroglioma and mixed glioma, with progressive disease on MRI,
were eligible for the study. TMZ was delivered at the starting dose of 200
mg/m
2
/day for 5 days, repeated monthly. Response was evaluated by MRI
(T2FLAIR weighted sequences). Deletion on 1p was searched by the LOH
technique. From 1999 to 2004, 99 consecutive patients were included in
the study (median age, 44 years; median Karnofsky score, 90). Median
follow-up was 22 months (654), and the median number of TMZ cycles
was 18 (626). The response rates were as follows: partial response (PR),
25%; minor response (MR), 14%; stable disease (SD), 52%; and progres-
sive disease (PD), 9%, respectively. Grade 34 toxicity occurred in 6% of
patients. The median PFS was 31 months (95% CI, 19 months). Blood
and tumor DNA pairs from 50 patients were available for LOH analysis.
LOH 1p was present in 22 tumors (corresponding to 15 PR MR, 7 SD, 0
PD) and absent in 28 tumors (corresponding to 4 PR MR, 20 SD, 4 PD)
(P 0.0001). We conrm in a larger series that TMZ provides a substantial
rate of objective response in LGOT and that LOH 1p is closely correlated
with radiographic response.
470. PHASE 2 MULTICENTER STUDY OF DOSE-INTENSE
TEMOZOLOMIDE IN PATIENTS WITH NEWLY DIAGNOSED
PURE AND MIXED ANAPLASTIC OLIGODENDROGLIOMA
D. Peereboom,
1
C. Brewer,
1
D. Schiff,
2
P. Fisher,
3
S. Snodgrass,
4
R.
Prayson,
1
G. Stevens,
1
M. Vogelbaum,
1
S. Panullo,
5
and G. Barnett
1
;
1
Brain Tumor Institute, Cleveland Clinic Foundation, Cleveland, Ohio;
2
Department of Neurology, University of Virginia, Charlottesville,
Virginia;
3
Department of Neurology, Stanford University, Palo Alto,
California;
4
Department of Neuro-Oncology, H. Lee Moftt Cancer
Center, Tampa, Florida;
5
Department of Neuro-Oncology, Columbia
Medical College, New York, New York; USA
Standard initial therapy for patients with pure and mixed anaplastic
oligodendrogliomas (AO/MAO) has included chemotherapy and radiation
therapy. These gliomas have particular sensitivity to chemotherapy, which
varies according to the molecular genetics of the tumor. Due to their chemo
responsiveness, this trial has used a dose-intense regimen of temozolomide
and reserved RT for patients with disease progression. Our objectives were
to determine the progression-free survival, response rate, and quality of
life (QOL) in patients with newly diagnosed AO/MAO treated with temo-
zolomide every other week and to determine outcomes according to tumor
cytogenetic status. Eligible patients had newly diagnosed AO/MAO with no
prior chemotherapy or radiation therapy. All pathology had central review
and tumor assay for 1p deletion using FISH (uorescent in situ hybridiza-
tion). Analysis was stratied by 1p status. Temozolomide was given 150
mg/m
2
, days 17 and 1521, every 28 days. Therapy was given for up to 8
cycles in the absence of progressive disease. Responses and QOL (FACT-
BR and EORTC brain module) were measured every 8 weeks. To date,
41 patients have been enrolled from 7 centers. Four patients are too early
to evaluate. Median age is 42 (range, 2083); Karnofsky PS, 100 in 10
pts, 90 in 16 pts, 80 in 3 pts, and 70 in 7 pts. Histology: AO in 21 pts,
MAO in 15 pts, and 1p deletion in 21 pts (15/21 with AO and 6/15 with
MAO). Patients have received 0 to 8 cycles (median 8) of temozolomide
(21 patients have completed all 8 cycles, 7 withdrew consent for toxicity or
patient choice prior to completion, 5 withdrew for progressive disease prior
to completion, 2 continue with stable disease in cycle 6, 1 withdrew prior to
receiving treatment). Only 1 patient has required dose reduction for toxicity
(20% reduction for thrombocytopenia). Nineteen patients remain free from
progression with a median progression-free survival of 10 months (range,
225). The overall survival is 14 months (534). Of the 32 patients with
response data available, 2 (AO with 1p loss) (6%) achieved complete remis-
sion, 17 (53%) have stable disease, and 13 (41%) had progression. Toxicity
was as follows: Grade 3 and 4 toxicities have included 6 pts with grade 3/4
neutropenia, 3 with grade 3 thrombocytopenia, and 11 with grade 3 lym-
phopenia; grade 1 and 2 toxicities have included fatigue, nausea, vomiting
and constipation. QOL data will be presented upon study completion. This
trial supports the concept of using chemotherapy alone as initial therapy for
patients with chemotherapy-responsive gliomas. The durability of responses
is not yet known. Temozolomide offers a reasonable alternative to initial RT
for patients with newly diagnosed AO/MAO. Toxicity in this trial has been
manageable. Accrual continues.
471. TEMOZOLOMIDE (TMZ) WITH O6-BENZYLGUANINE
(BG) FOR TMZ-RESISTANT MALIGNANT GLIOMA: A
PHASE 2 TRIAL
A. Desjardins, J. Quinn, D. Reardon, J. Rich, S. Sathornsumetee, J.
Vredenburgh, A. Walker, S. Tourt-Uhlig, D. Bigner, and H. Friedman;
Duke University Medical Center, Durham, North Carolina, USA
TMZ is a methylating agent with conrmed activity in recurrent and
newly diagnosed malignant glioma. One of the major mechanisms of resis-
tance to TMZ is determined by the DNA repair protein O6-alkylguanine-
DNA alkyltransferase (AGT), which removes the methylation damage from
O6 position of guanine. BG inhibits AGT by acting as an AGT substrate.
We performed a phase 2 study of TMZ combined with BG in patients with
TMZ-resistant malignant glioma. Eligibility included adult patients with
malignant gliomas resistant to TMZ. Patients were divided in two strata,
one for glioblastoma multiforme (GBM) and the second for anaplastic
astrocytoma or oligodendroglioma (AA/AO). Each patient was treated
with 1-h BG infusion at 120 mg/m, followed by a single dose of TMZ at
472 mg/m, and thereafter a 48-h continuous BG infusion at 30 mg/m/day.
Responses were assessed by functional and imaging criteria after two cycles
(8 weeks). Patients were treated for one year or until disease progression or
unacceptable toxicity. The primary end points of this study were to dene
the role of BG in restoring TMZ sensitivity in patients with TMZ-resis-
tant malignant glioma and to determine the activity and toxicity of TMZ
plus BG. To date, 62 patients with recurrent malignant gliomas (GBM
34, AA/AO 28) have been enrolled, for a planned accrual of 64 patients.
Fifty-ve patients are assessable for response. Five patients showed a par-
tial response (AA/AO n 4, GBM n 1), 17 showed a stable disease, and
33 patients progressed at the 6-week evaluation. Toxicities are limited to
hematologic toxicity and included neutropenia (36 grade 4, no grade 3),
leukopenia (one grade 4), thrombocytopenia (8 grade 4), anemia (one grade
3, one grade 4) and one case of febrile neutropenia. Toxicities are limited
to hematologic events. Activity is seen in TMZ refractory AA/AO and war-
rants further study. Trivial activity is seen in TMZ refractory GBM, and the
reasons may include suboptimal TMZ schedule, alternative mechanisms of
resistance, and AGT mutations.
472. A PHASE 2 STUDY OF TEMOZOLOMIDE (TMZ)
AND THE FARNESYLTRANSFERASE INHIBITOR (FTI)
LONAFARNIB (SARAZARTM, SCH66336) IN RECURRENT
GLIOBLASTOMA
M. Gilbert,
1
K. Hess,
1
V. Liu,
1
M. Groves,
1
V. Puduvalli,
1
V. Levin,
1

C. Conrad,
1
H. Colman,
1
S. Hsu,
1
M.M. Sugrue,
2
and W.K.A.
Yung
1
;
1
Neuro-oncology, The University of Texas M.D. Anderson
Cancer Center, Houston, Texas;
2
Schering-Plough Research Institute,
Kennilworth, New Jersey; USA
Farnesylation is an essential step in the post-translational modication
of several proteins that play a role in cell proliferation and growth, includ-
ing Ras, RhoB, centromere binding proteins (CENP-E/CENP-F), lamin B,
and protein tyrosine phosphatase (PTP). Inhibition of farnesylation may
inhibit tumor cell proliferation. Preclinical testing demonstrates antiprolif-
erative effects of FTIs in a variety of tumor cell lines and xenograft models.
Additional laboratory data from animal models demonstrated enhanced
efcacy of combining lonafarnib with TMZ compared with TMZ alone.
Our objective was to determine the efcacy as measured by response rate
and 6-month progression-free survival rate of the combination of TMZ
with lonafarnib. Eligibility criteria were as follows: histologically proven
GBM, unequivocal evidence of tumor relapse or progression after radiation
therapy, up to 2 prior chemotherapy regimens for recurrent disease, ability
to provide informed consent, and KPS of 60. Patients may not be on cyto-
chrome P450-inducing anticonvulsants. The treatment plan was as follows:
TMZ was administered at 150 to 200 mg/m
2
days 15 and lonafarnib at
150 mg BID on days 828 of a 28-day cycle. Response was evaluated every
2 cycles. Standard response criteria established by MacDonald were used.
Twenty-three patients were accrued to the study. Average age was 53 years
and median KPS was 90. Male to female ratio was 15:8. Overall, 10 patients
demonstrated stable disease (including 3 minor responses), and 3 patients
had a partial response. The 6-month PFS rate was 13%, median PFS was
16 weeks, and overall survival from study entry was 38 weeks. Treatment
was well tolerated. One patient had grade 3 granulocytopenia, thrombo-
cytopenia, and anemia, one patient had grade 4 granulocytopenia, and one
patient had grade 4 thrombocytopenia. One patient developed grade 3 diar-
rhea. The combination of TMZ with lonafarnib using a conventional TMZ
dosing schedule was well tolerated, but these data do not support improved
efcacy over TMZ alone. Recent reports suggest that the dose-density of
TMZ can be safely increased, and this may enhance activity. Additionally,
modifying the dose and schedule of the lonafarnib may improve efcacy
when combined with the dose-intensied TMZ regimen. A phase 1 study
is under way to optimize the TMZ-lonafarnib combination.
473. PHASE 2 TRIAL OF VINCRISTINE, NIMUSTINE
(ACNU), CARBOPLATIN, AND INTERFERON-BETA WITH
RADIATION THERAPY (VAC-FERON-R ) FOR NEWLY
DIAGNOSED GLIOBLASTOMA MULTIFORME
T.A. Aoki,
1
J.A. Takahashi,
2
T.U. Ueba,
2
K.N. Nozaki,
2
N.O.
Oya,
3
M.H. Hiraoka,
3
A.N. Nakajima,
4
K.M. Matsui,
5
T.F. Fukui,
5

M.I. Ishikawa,
1
and N.H. Hashimoto
2
;
1
Neurosurgery, Osaka;
2
Neurosurgery, Kyoto;
3
Radiation Oncology, Kyoto;
4
Pathology,
Kyoto;
5
Kyoto University Hospital, Biostatics, Kyoto; and the Kyoto
Neuro-Oncology Group (KNOG): Kyoto University, Kitano Hospital,
Shimizu Hospital, Kyoto National Hospital, Himeji National Hospital,
Tenri Yorozu Hospital, Takeda Hospital, Osaka Red Cross Hospital,
Kishiwada City Hospital, Fukui Red Cross Hospital, Kurasiki Central
Hospital, National Cardio-Vascular Center, Saiseikai Noe Hospital,
Kizugawa Hospital, Hikone City Hospital, Shizuoka Preferectual
Hospital, Saisekai Nakatsu Hospital, Hamamatsu Rousai Hospital,
Tsukaguchi Preferectual Hospital, Kobe Metal Hospital, Nagahama City
Hospital, Takatsuki Red Cross Hospital, Kobe Central City Hospital;
Japan
Novel combination chemotherapies have been needed for patients with
glioblastoma multiforme, because single drugs have not been sufciently
effective. Based on previous published reports, we select four drugs (vin-
cristine, ACNU, carboplatin, and interferon-beta). This phase 2 study was
performed to determine the safety, tolerability, and efcacy for this therapy,
after a dose setting by our feasibility study. This multicenter phase 2 trial
enrolled 97 patients (intent-to-treat population). After central histologic
review, 97 patients were conrmed to have had a GBM. ACNU (60 mg/
m
2
), carboplatin (110 mg/m
2
), vincristine (0.6 mg/m
2
) and interferon-beta
(300 million/body) were administered in day 1, concomitant with fraction-
ated radiotherapy (63 Gy total dose; 1.8 Gy 5 days for 7 cycles) and
vincristine (0.6 mg/m
2
) and interferon-beta (300 million/body) in day 7
and 15, interferon-beta (300 million/body) three times for a week during
the radiation course. Two months after the radiation, ACNU (60 mg/m
2
),
carboplatin (110 mg/m
2
), vincristine (0.6 mg/m
2
) and interferon-beta (300
million/body) were administered in day 1 and vincristine (0.6 mg/m
2
) and
interferon-beta (300 million/body) in day 7 and 15, every 58 days. The pri-
mary end points were safety and tolerability, and the secondary end points
were time to progression and overall survival. Nonhematologic toxicities
were rare and mild to moderate in severity. During the radiation treatment
phase, grade 3 toxicities in neurocytopenia, or thrombocytopenia, or both
were observed in 23% of patients. Grade 4 hematological toxicities were
not observed. During adjuvant maintenance chemotherapy after radiation,
7% of all cycles were associated with grade 3 toxicities in neurocytopenia
or thrombocytopenia. None of the cycles were with grade 4 toxicities. Time
to progression (TTP) was 11 months, and median survival was 16 months.
VAC-feron-R is safe and well tolerated. This regimen of concomitant
chemo-radiation therapy followed by adjuvant maintenance chemotherapy
may prolong the survival of patients with GBM. Further investigation is
warranted.
Neuro-Oncology 7, 279402, 2005 (Posted to Neuro-
Oncology [serial online], Doc. 05-2038, June 17, 2005.
URL http://neuro-oncology.mc.duke.edu; DOI: 10.1215/
S1152851705200388)

Abstracts From The World Federation of Neuro-Oncology Second Quadrennial Meeting and The Sixth Meeting of The European Association For Neuro-Oncology

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